This study examined the binding interactions between a natural MUC1 peptide epitope (GVTSAPD) and 6 derivative epitopes with a MUC1 monoclonal antibody (mAb 6A4) using Saturation Transfer Difference (STD) NMR spectroscopy. The results showed that the proline residue at position 6 of the natural epitope is critical for antibody binding. Substituting this proline for aromatic residues like tryptophan and phenylalanine maintained some binding ability, while substitution with aliphatic or polar residues eliminated binding. These findings help identify residues important for antibody recognition and could inform the design of cancer vaccine candidates.
The Matrix metalloproteinase-9 is involved in several pathologies. Its strong presence in ocular pathologies explains our interest for its genetic variation in cataract, glaucoma and retinoblastoma in Senegal. MMP9 is highly polymorphic with cataract and glaucoma. 77 mutations were noted with 21 haplotypes for the entire population. The haplotype diversity Hd is 0.831 and the nucleotide diversity Pi is 0.016; k = 17.395. The polymorphism of the Matrix metalloproteinase-9 gene is associated with all three diseases and SNP 3918249 is found in both cataract and glaucoma.
This is Part 2 of a presentation on Genetic Toxicology that was given by Dr. David Kirkland to scientific staff at Health Canada in Nov. 2010. Part 1 is availabile here in ppt and as a webinar at the LinkedIn DABT CE group link
Targeting PIM kinase to overcome drug resistance in NSCLC - Dr Kathy GatelyHannahMcCarthy31
Dr Kathy Gately is a Clinical Scientist at St James's Hospital and Clinical Senior Lecturer at Trinity College Dublin. Dr Gately's research interests are Drug Resistance Mechanisms in Non-Small Cell Lung Cancer, Liquid Biopsy and Organoid models.
The Matrix metalloproteinase-9 is involved in several pathologies. Its strong presence in ocular pathologies explains our interest for its genetic variation in cataract, glaucoma and retinoblastoma in Senegal. MMP9 is highly polymorphic with cataract and glaucoma. 77 mutations were noted with 21 haplotypes for the entire population. The haplotype diversity Hd is 0.831 and the nucleotide diversity Pi is 0.016; k = 17.395. The polymorphism of the Matrix metalloproteinase-9 gene is associated with all three diseases and SNP 3918249 is found in both cataract and glaucoma.
This is Part 2 of a presentation on Genetic Toxicology that was given by Dr. David Kirkland to scientific staff at Health Canada in Nov. 2010. Part 1 is availabile here in ppt and as a webinar at the LinkedIn DABT CE group link
Targeting PIM kinase to overcome drug resistance in NSCLC - Dr Kathy GatelyHannahMcCarthy31
Dr Kathy Gately is a Clinical Scientist at St James's Hospital and Clinical Senior Lecturer at Trinity College Dublin. Dr Gately's research interests are Drug Resistance Mechanisms in Non-Small Cell Lung Cancer, Liquid Biopsy and Organoid models.
Immunologic tumour models in NSCLC - Dr Catriona DowlingHannahMcCarthy31
Dr Dowling is a lecturer in biomedical sciences and is a lung cancer researcher at the University of Limerick. Dr Dowling's special research interests are immuno-metabolism and basic science in lung cancer.
Austin Journal of Nanomedicine & Nanotechnology is an open access, peer reviewed, scholarly journal dedicated to publish innovative research works carried out in the fields of Nanomedicine & Nanotechnology.
Austin Journal of Nanomedicine & Nanotechnology aims to serve health care professionals, medical practitioners and innovative researchers by providing a forum to find most recent advances in the areas Nanomedicine & Nanotechnology.
Austin Journal of Nanomedicine & Nanotechnology accepts original research articles, review articles and short communication on all the aspects of Nanomedicine & Nanotechnology for review and possible publication.
Austin Journal of Nanomedicine & Nanotechnology is an open access, peer reviewed, scholarly journal dedicated to publish innovative research works carried out in the fields of Nanomedicine & Nanotechnology.
Assessment of immunomolecular_expression_and_prognostic_role_of_tlr7_among_pa...dr.Ihsan alsaimary
Dr. ihsan edan abdulkareem alsaimary
PROFESSOR IN MEDICAL MICROBIOLOGY AND MOLECULAR IMMUNOLOGY
ihsanalsaimary@gmail.com
mobile : 009647801410838
university of basrah - college of medicine - basrah -IRAQ
38th FEBS Congress
Maxim L. Bychkov
Poster for abstract: "DR5-B – DR5-selective mutant variant of cytokine TRAIL overcomes resistance of cancer cells to TRAIL"
Abstract book, p. 595
Annals of Mutagenesis is an open access, peer reviewed, scholarly journal dedicated to publish articles covering all areas of Mutagenesis.
The journal aims to promote research communications and provide a forum for doctors, researchers, physicians and healthcare professionals to find most recent advances in all areas of Mutagenesis. Annals of Mutagenesis accepts original research articles, reviews, mini reviews, case reports and rapid communication covering all aspects of mutagenesis.
Annals of Mutagenesis strongly supports the scientific up gradation and fortification in related scientific research community by enhancing access to peer reviewed scientific literary works. Austin Publishing Group brings universally peer reviewed journals under one roof thereby promoting knowledge sharing, mutual promotion of multidisciplinary science.
Immunologic tumour models in NSCLC - Dr Catriona DowlingHannahMcCarthy31
Dr Dowling is a lecturer in biomedical sciences and is a lung cancer researcher at the University of Limerick. Dr Dowling's special research interests are immuno-metabolism and basic science in lung cancer.
Austin Journal of Nanomedicine & Nanotechnology is an open access, peer reviewed, scholarly journal dedicated to publish innovative research works carried out in the fields of Nanomedicine & Nanotechnology.
Austin Journal of Nanomedicine & Nanotechnology aims to serve health care professionals, medical practitioners and innovative researchers by providing a forum to find most recent advances in the areas Nanomedicine & Nanotechnology.
Austin Journal of Nanomedicine & Nanotechnology accepts original research articles, review articles and short communication on all the aspects of Nanomedicine & Nanotechnology for review and possible publication.
Austin Journal of Nanomedicine & Nanotechnology is an open access, peer reviewed, scholarly journal dedicated to publish innovative research works carried out in the fields of Nanomedicine & Nanotechnology.
Assessment of immunomolecular_expression_and_prognostic_role_of_tlr7_among_pa...dr.Ihsan alsaimary
Dr. ihsan edan abdulkareem alsaimary
PROFESSOR IN MEDICAL MICROBIOLOGY AND MOLECULAR IMMUNOLOGY
ihsanalsaimary@gmail.com
mobile : 009647801410838
university of basrah - college of medicine - basrah -IRAQ
38th FEBS Congress
Maxim L. Bychkov
Poster for abstract: "DR5-B – DR5-selective mutant variant of cytokine TRAIL overcomes resistance of cancer cells to TRAIL"
Abstract book, p. 595
Annals of Mutagenesis is an open access, peer reviewed, scholarly journal dedicated to publish articles covering all areas of Mutagenesis.
The journal aims to promote research communications and provide a forum for doctors, researchers, physicians and healthcare professionals to find most recent advances in all areas of Mutagenesis. Annals of Mutagenesis accepts original research articles, reviews, mini reviews, case reports and rapid communication covering all aspects of mutagenesis.
Annals of Mutagenesis strongly supports the scientific up gradation and fortification in related scientific research community by enhancing access to peer reviewed scientific literary works. Austin Publishing Group brings universally peer reviewed journals under one roof thereby promoting knowledge sharing, mutual promotion of multidisciplinary science.
Proteogenomic analysis of human colon cancer reveals new therapeutic opportun...Gul Muneer
We performed the first proteogenomic study on a prospectively collected colon cancer cohort. Comparative proteomic and phosphoproteomic analysis of paired tumor and normal adjacent tissues produced a catalog of colon cancer-associated proteins and phosphosites, including known and putative new biomarkers, drug targets, and cancer/testis antigens. Proteogenomic integration not only prioritized genomically inferred targets, such as copy-number drivers and mutation-derived neoantigens, but also yielded novel findings. Phosphoproteomics data associated Rb phosphorylation with increased proliferation and decreased apoptosis in colon cancer, which explains why this classical tumor suppressor is amplified in colon tumors and suggests a rationale for targeting Rb phosphorylation in colon cancer. Proteomics identified an association between decreased CD8 T cell infiltration and increased glycolysis in microsatellite instability-high (MSI-H) tumors, suggesting glycolysis as a potential target to overcome the resistance of MSI-H tumors to immune checkpoint blockade. Proteogenomics presents new avenues for biological discoveries and therapeutic development.
Aptamer Technology in the Targeted Delivery of Doxorubicin | Crimson PublishersCrimsonpublishersCancer
This review aims at giving an insight at some of the recent approaches in using the aptamer technology in formulating the chemotherapeutic agent, doxorubicin, so as to achieve its targeted delivery and reduce its non-specific cytotoxicity.
PTPRC as a Predictive Marker Related to PD-L1 for Prognosis and Immunotherapy...semualkaira
The expression of programmed cell death protein 1 (PD-L1) has been found to be closely related to the efficacy
of immunotherapy. The aim of our study is to explore biomarkers
associated with PD-L1 expression that might influence the efficacy
of immunotherapy.
PTPRC as a Predictive Marker Related to PD-L1 for Prognosis and Immunotherapy...semualkaira
The expression of programmed cell death protein 1 (PD-L1) has been found to be closely related to the efficacy of immunotherapy. The aim of our study is to explore biomarkers associated with PD-L1 expression that might influence the efficacy of immunotherapy.
PTPRC as a Predictive Marker Related to PD-L1 for Prognosis and Immunotherapy...semualkaira
The expression of programmed cell death protein 1 (PD-L1) has been found to be closely related to the efficacy of immunotherapy. The aim of our study is to explore biomarkers associated with PD-L1 expression that might influence the efficacy of immunotherapy.
ShRNA-specific regulation of FMNL2 expression in P19 cellsYousefLayyous
This video encompasses all the steps and data produced for my graduation project in BSc in Biopharmaceutical science. During the course of the project we modified mammalian cells using Short Hairpin RNA to inhibit the correct function of the cytoskelleton. In this way we studied the importance of FMNL2 for the activation and regulation of actin fibers. Among the methods used are Flourescent microscopy, mamallian cell culture, cloning and flow cytometry.
1. Mass Spectral Analysis
Mass spectra were acquired to begin characterization of obtained peptide
fractions. These spectra were compared to the theoretical masses of each
peptide epitope used in the study.
NMR Spectroscopy
2D TOCSY and ROESY 1H NMR were used to complete unambiguous
proton assignments for all peptide epitopes. STD NMR technique was
used to determine peptide-antibody binding. Fig. 4 below shows one such
spectrum off the peptide epitope GVTSAWD. [10,11].
Fig. 4. Overlay of TOCSY (green) and ROESY (red) NMR spectra of peptide epitope
GVTSAWD.
Mucins (shown in Fig. 1) are a class of heavily o-glycosylated proteins,
most commonly found on epithelial surfaces, which provide many
protective cellular functions such as the formation of mucosal barriers [1].
The range of human mucins (MUC) spreads from MUC1 to MUC21,
however the specific mucin this study is associated with is the MUC1
transmembrane protein. MUC1 has been detected as a carcinoma-linked
antigen. This is believed to be caused by a loss in cellular polarity
attributed in part to MUC1 protein. This loss of polarity triggers an
epithelial-mesenchymal transition (EMT) producing aggressive cancer
cells. Additionally, transmembrane mucins have been found to be
overexpressed in cancer cells, leading to prolonged EMT activation and
eventual malignancy [2,3]. Thus far it has been difficult to create an
effective immunotherapy or vaccine for carcinomas as many tumors
produce immunosuppressive effects [5,6].
It is believed that using truncated MUC1 peptide sequences could be an
effective vaccination against carcinomas due to the nature of the o-
glycosylation of cancer cell-associated mucins. These mucin proteins are
overexpressed and hypoglycosylated, leaving short amino acid sequences
exposed to the extracellular environment [4,5,7]. This potentially increases
the efficacy of these proteins as antigens for the signaling of cytotoxic T
cells if previously exposed to a vaccination of short peptide epitopes of
MUC1 [4,5]. Furthermore, evidence shows that using a mimotope, a
derivative sequence of the exposed peptide epitope (a substitution of 1 or 2
amino acids), may elevate the immune response when presented with a
tumor cell exhibiting the natural epitope [5,6].
Fig. 1 Transmembrane mucin protein with o-glycosylation explicitly shown. This
glycosylation is less dense in mucins associated with cancer cell membranes [12].
This study examines the antibody-binding efficacy of 6 of these MUC1
epitope derivatives in addition to the natural epitope that appears in the
previously studied tandem repeat GVTSAPDTRPAPGSTAPPAH [7,8].
This was done via saturation transfer difference (STD) NMR with the
purpose of determining the specific residues that play an active role in
antibody binding. Additionally, the study aims to identify a derivative
epitope that would exhibit strong antibody-binding interactions that would
eventually elicit a more efficient immune response. Results are shown in
Fig 3: panels A and B.
Abstract Objectives Methods
Conclusions
References
Analysis of the Antibody Binding of Derivative MUC1 Peptides via STD NMR
Andrew R. Lynch and Thao Yang
Department of Chemistry, University of Wisconsin-Eau Claire
249th ACS National Meeting & Exposition, March 22nd – 26th, 2015
The results indicate that Proline-substituted derivatives GVTSAWD and
GVTSAFD retain the ability to bind mAb(6A4), but to a much lesser
degree; GVTSALD and GVTSADD do not, however. The normal epitope
GVTSAPD, upstream from the main binding site in its domain, retained
mAb binding ability as well and was further maintained when Ser4 was
replaced with Asn (GVTNAPD), indicating that Pro6 is critical for
antibody binding. This information indicates that hydrophobic ring
structures such as Trp, Phe and Tyr at the Pro position are critical for the
binding of antibody.
1. Kufe, D. W. Mucins in cancer: function, prognosis and therapy. Nature
Reviews: Cancer. 9, 874-885 (2009).
2. Oosterkamp, H.M., Scheiner, L., Stefanova, M.C., Lloyd, K. O. and
Finstad, C.L. Comparison of MUC-1 mucin expression in epithelial
and non-epithelial cancer cell lines and demonstration of a new short
variant form (MUC-1/Z). Int. J. Cancer, 72: 87-94 (1997).
3. Kalluri, R., & Weinberg, R. The Basics Of Epithelial-mesenchymal
Transition. Journal of Clinical Investigation, 119(6), 1420-1428 (2009).
4. Roulois, D., Grégoire, M., & Fonteneau, J. MUC1-specific cytotoxic T
lymphocytes in cancer therapy: induction and challenge. Biomed
Research International, 2013871936 (2013).
5. Buhrman, J., & Slansky, J. Improving T cell responses to modified
peptides in tumor vaccines. Immunologic Research, 55(0), 34-47
(2013).
6. Laing, P., Tighe, P., Kwiatkowski, E., Milligan, J., Price, M., & Sewell,
H. Selection of peptide ligands for the antimucin core antibody C595
using phage display technology: Definition of candidate epitopes for a
cancer vaccine. Molecular Pathology, (48), M136-M141 (1995).
7. A.-B.M. Abdel-Aal, V. Lakshminarayanan, P. Thompson, N. Supekar,
J.M. Bradley, M.A. Wolfert, P.A. Cohen, S.J. Gendler, and G.-J. Boons.
ChemBioChem., 15, 1508-1513 (2014).
8. N. Gaidzik, U. Westerlind, and H. Kunz. Chem. Soc. Rev., 4, 4421-
4442 (2013)
9. W.C. Chan, and P.D. White. Basic Procedures in: Fmoc Solid Phase
Peptide Synthesis, A Practical Approach. Oxford, New York: Oxford
University Press, p. 41-76 (2000).
10. Her, C., and Yang, T., Antibody Binding Study of Mucin Peptide
Epitopes. Division of Biological Chemistry, 243rd ACS meeting, San
Diego, CA., March 25-29, 2012.
11. C. Her, W.M. Westler, and T. Yang. JSM Chem. 1 (1), 1004 (2013).
12. "MUCIN." Sigma Aldrich. Sigma-Aldrich Co. LLC, n.d. Web.
<http%3A%2F%2Fwww.sigmaaldrich.com%2Flife-
science%2Fmetabolomics%2Fenzyme-explorer%2Flearning-
center%2Fstructural-proteins%2Fmucin.html>.
I would like to thank the UWEC Office of Research and Sponsored
Programs (ORSP) for providing the Faculty/Student Collaborative and
travel grants to support this research, the Chemistry Department for use of
the NMR spectrometer, and also the Blugold Commitment for printing this
poster. In addition I would also like to thank the ACS and abstract review
board for holding this exposition.
Results
Multivalency MUC1 antigens are being sought in the development of
cancer vaccine. Substituted mucin peptides such as GVTSAFD and
GVTSAWD and their derivatives (cyclic mucin peptides) that have binding
ability to MUC1 antibody may provide useful additional epitopes toward
designing such agents.
The purpose of the study is to examine the strength of binding interactions
between one natural MUC1 epitope and 6 derivative epitopes with
monoclonal antibody (mAb) 6A4 via STD NMR. Fig. 2 shows a
representation of the natural epitope. Epitopes used are as follows:
Fig. 2 Representation of natural epitope GVTSAPD.
Natural Peptide Epitope:
GVTSAPD
Derivative peptide epitopes used in this study: *
GVTNAPD
GVTSADD
GVTSALD
GVTSAYD
GVTSAFD
GVTSAWD
*Bolded characters indicate amino acid deviations from the natural epitope.
N
O
O
O
N
O
N
O
N
O
O
N
O
O
N
O
N
O
A B
Fig 3: Panel A Pairs of 1H STD NMR spectra (bottom) and reference 1H NMR spectra
(top) in the presence of mAb (6A4) in 20 mM phosphate, 5 mM NaCl, pH 7.0, 100% D2O, 7
°C. Dashed lines connect the protons that experienced STD effect on the STD NMR spectra
to the corresponding protons on the 1H NMR spectra. The pairs of spectral traces are: A&B).
for GVTSAPD peptide, with highest STD peak intensity located at Pro6Hδ resonance, its
intensity was adjusted to the same height as the one in the normal 1H NMR spectrum; note
that contaminants at 3.6 ppm were subtracted out in the STD spectra as they do not interact
with the mAb; C&D). for GVTNAPD peptide, with similar STD effects to spectrum (A);
E&F). for GVTSAWD peptide; G&H). for GVTSAFD peptide. Contaminants are marked
by asterisks (*) on both panels. Peptide epitopes GVTSAPD, GVTSAWD, GVTNAPD, and
GVTSAFD were all shown to bind mAb (6A4) though at differing intensities.
Fig 3: Panel B Pairs of 1H STD NMR spectra (bottom) and reference 1H NMR
spectra (top) in the presence of mAb. The pairs of spectral traces are: I&J). for
GVTSADD peptide, showing flat line for STD NMR spectrum; K, L&M). for GVTSAYD
peptide; (K), same spectrum as L but without protein background subtraction; N&O). for
GVTSALD. Peptide epitopes GVTSADD and GVTSALD were not shown to bind mAb
(6A4). Peptide epitope GVTSAYD, however, did display limited binding capability.
Introduction
The binding epitope PDTRP, found within the VNTR domain of MUC1
glycoprotein, is recognized by the immune system and binds mucin
monoclonal antibody SM3. This study analyzes the binding ability of the
peptide GVTSAPD, an upstream sequence preceding PDTRP, as well as
six derivative peptides against specific mouse MUC1 mucin monoclonal
antibody (IgG1, 6A4) by Saturation Transfer Difference NMR (STD NMR)
to determine the specific residue critical for antibody binding and whether
analogous side chain characteristics in the derivative sequences would
enhance binding. Of the derivative sequences resembling epitope
GVTSAPD, five had the Pro6 residue substituted for Asp, Leu, Tyr, Phe,
and Trp and one had the Ser4 residue substituted for Asn. The STD NMR
results indicated that Pro6 is a critical residue for binding as it displayed
greater saturation transfer effects for its side chain protons than did any
other residue. Substituting the Pro6 residue with single hydrophilic or
hydrophobic aliphatic residue (Leu or Asp) eliminated all STD effects
while substitution of the same residue with single hydrophobic aromatic
residues (Phe or Trp) produced STD effects at the aromatic protons.
Substitution at Ser4 position for Asn produced STD effects that were rather
similar in pattern and intensity to those of the native sequence. As indicated
by the results, the Pro6 residue on native sequence, GVTSAPD, Pro6 in
particular, is critical for antibody binding and substitution at this position
for aromatic residues (Trp or Phe) conserves binding ability, which
suggests that these substituted peptides may possess biological activity.
Methods
Epitope Modification via Peptide Synthesis
All peptides used in this study were manually synthesized in the solid-
phase using standard Fmoc-chemistry [9].
HPLC Purification
Purification of synthesized peptides was carried out via reverse phase
HPLC with a C18 column and water and acetonitrile mobile phases.
Isolated peptide fractions were collected for further analyses using a
UV/Visible light detection module. Results seen in Fig. 5.
Acknowledgements
Results
Fig. 5. HPLC spectrum showing isolated peak of peptide epitope GVTSAWD.