Sortases are bacterial enzymes capable of protein transacylation, using a wide variety of donor and acceptor molecules. The SrtA class of sortase recognizes an LPXTG sequence in a suitably engineered protein substrate or in a synthetic peptide sequence, which it cleaves between the Thr and Gly residues with concomitant formation of an acyl-enzyme intermediate. This intermediate is then resolved by nucleophilic attack, using synthetic primary amines or proteins suitably equipped with Gly or Ala as the N- terminus. Using sortases as tools, new protein labeling strategies will be developed that enable protein modifications not attainable by genetic means, such as the C-terminus to C-terminus fusion of two distinct polypeptides. These methods will be applied to the synthesis of recombinant proteins of immunological interest such as Class II MHC products, T cell receptor ectodomains and antibody F(ab) fragments, with a view to create labeled versions of these proteins that can be used for detection and isolation of their relevant counterstructures, or to enumerate the cells that bear receptors for them. The production of Class II MHC tetramers remains cumbersome, and the proposed methods have the potential of dramatically simplifying the production of these key diagnostic tools used to track pathogenic and protective T cell responses alike. The range of substrates that can be modified will be extended through the development of orthogonal labeling strategies that employ sortases of different specificities, either in their peptide recognition sequence or in their ability to accept certain types of nucleophile. This will be accomplished not only through site-directed mutagenesis of the Srt A enzymes of Staphylococcus aureus and Streptococcus pyogenes, but also through the use of other classes of sortases (e.g. SrtB from S. aureus or B. anthracis). Finally, we shall apply this technology to the question of flu particle biogenesis, a process that has so far defied observation in real time, but that may be visualized using the labeling strategies proposed here as a possible means to identify discrete steps that might serve as targets for intervention. The significance of the proposed studies lies in the development of methods that will enable the site-specific modification of proteins with entities that cannot be installed genetically.

Public Health Relevance

New chemoenzymatic methods will be developed to facilitate the generation of diagnostic tools that can be used to track immune responses that protect against infectious agents as well as those that cause autoimmunity. Similar protein modification strategies will be applied to study how flu virus particles are assembled and released from the infected cell. The proposed combination of chemistry and biology will generate new possibilities for the diagnosis and treatment of disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI087879-05
Application #
8627535
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Huntley, Clayton C
Project Start
2010-03-15
Project End
2015-02-28
Budget Start
2014-03-01
Budget End
2015-02-28
Support Year
5
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Whitehead Institute for Biomedical Research
Department
Type
DUNS #
City
Cambridge
State
MA
Country
United States
Zip Code
02142
Rashidian, Mohammad; Ingram, Jessica R; Dougan, Michael et al. (2017) Predicting the response to CTLA-4 blockade by longitudinal noninvasive monitoring of CD8 T cells. J Exp Med 214:2243-2255
Van Elssen, Catharina H M J; Rashidian, Mohammad; Vrbanac, Vladimir et al. (2017) Noninvasive Imaging of Human Immune Responses in a Human Xenograft Model of Graft-Versus-Host Disease. J Nucl Med 58:1003-1008
Wu, Qin; Ploegh, Hidde L; Truttmann, Matthias C (2017) Hepta-Mutant Staphylococcus aureus Sortase A (SrtA7m) as a Tool for in Vivo Protein Labeling in Caenorhabditis elegans. ACS Chem Biol 12:664-673
Ingram, Jessica R; Blomberg, Olga S; Sockolosky, Jonathan T et al. (2017) Localized CD47 blockade enhances immunotherapy for murine melanoma. Proc Natl Acad Sci U S A 114:10184-10189
Antos, John M; Ingram, Jessica; Fang, Tao et al. (2017) Site-Specific Protein Labeling via Sortase-Mediated Transpeptidation. Curr Protoc Protein Sci 89:15.3.1-15.3.19
Dongre, Anushka; Rashidian, Mohammad; Reinhardt, Ferenc et al. (2017) Epithelial-to-Mesenchymal Transition Contributes to Immunosuppression in Breast Carcinomas. Cancer Res 77:3982-3989
Fang, Tao; Van Elssen, Catharina H M J; Duarte, Joao N et al. (2017) Targeted antigen delivery by an anti-class II MHC VHH elicits focused ?MUC1(Tn) immunity. Chem Sci 8:5591-5597
Pishesha, Novalia; Bilate, Angelina M; Wibowo, Marsha C et al. (2017) Engineered erythrocytes covalently linked to antigenic peptides can protect against autoimmune disease. Proc Natl Acad Sci U S A 114:3157-3162
Ingram, Jessica R; Dougan, Michael; Rashidian, Mohammad et al. (2017) PD-L1 is an activation-independent marker of brown adipocytes. Nat Commun 8:647
Hanke, Leo; Schmidt, Florian I; Knockenhauer, Kevin E et al. (2017) Vesicular stomatitis virus N protein-specific single-domain antibody fragments inhibit replication. EMBO Rep 18:1027-1037

Showing the most recent 10 out of 53 publications