Synthetic peptides can be used to investigate the effect of post- translational modifications on a wide range of biochemical, biophysical and biological processes In the first tow cycles of this grant, we have developed new methodologies for the solid-phase synthesis of glycopeptides and have established the principle effects glycosylation exerts on the conformational, in vitro stability and certain biological recognition properties of the peptide backbone. However, the examples provided by us and others to date are limited in size and complexity. In the current proposal, we will undertake the study of glycopeptides in more complex systems. We will prepare and use synthetic antibacterial glycopeptides, which have hitherto not been available in useful quantities to fully characterize their range of activity and mode of action. Pathobiochemical processes that are due to possible abnormal hyperglycosylation of potential N- glycosylation sites of proteins will also be studies with glycopeptides corresponding to fragments of the human tau protein, the main proteinaceous component of the neurofibrillary tangles of Alzheimer~s disease. Differentially glycosylated versions of the repeat unit of the mucin protein will serve as models of glycoprotein fragments on which the sugar length-dependent recognition of monoclonal antibodies and T-cells will be characterized. To this end, N-terminally protected, maltotriose-and maltoheptose-coupled serine and threonine residues will be chemically prepared. These derivatives will also be used as building blocks for middle-sized and long sugar containing T helper cell epitopic peptide models to study the binding of the glycopeptides to major histocompatibility proteins (MHC) and the T-cell receptor. Finally, a possible biotechnological use of glycopeptides will be assessed using mannosylated T-cell epitopes, in which the sugar appendages are expected to help deliver the epitopes to the intracellular compartments of antigen presenting cells. The above listed studies will be complemented with analysis of the secondary structure and stability of the glycopeptides to address specific questions concerning the mode and duration of action, and the sugar length-and anomeric configuration-dependent conformational transitions. The models and methods of this grant proposal can be used later as prototypes for studying a newer set of glycopeptide/glycoprotein functions, and may open doors to new ides and applications.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM045011-10
Application #
6180199
Study Section
Bio-Organic and Natural Products Chemistry Study Section (BNP)
Program Officer
Schwab, John M
Project Start
1990-06-05
Project End
2001-06-30
Budget Start
2000-07-01
Budget End
2001-06-30
Support Year
10
Fiscal Year
2000
Total Cost
$256,691
Indirect Cost
Name
Wistar Institute
Department
Type
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Cudic, M; Otvos Jr, L (2002) Intracellular targets of antibacterial peptides. Curr Drug Targets 3:101-6
Vlad, Anda M; Muller, Stefan; Cudic, Mare et al. (2002) Complex carbohydrates are not removed during processing of glycoproteins by dendritic cells: processing of tumor antigen MUC1 glycopeptides for presentation to major histocompatibility complex class II-restricted T cells. J Exp Med 196:1435-46
Cudic, Mare; Ertl, Hildegund C J; Otvos Jr, Laszlo (2002) Synthesis, conformation and T-helper cell stimulation of an O-linked glycopeptide epitope containing extended carbohydrate side-chains. Bioorg Med Chem 10:3859-70
Kragol, G; Otvos Jr, L; Feng, J et al. (2001) Synthesis of a disulfide-linked octameric peptide construct carrying three different antigenic determinants. Bioorg Med Chem Lett 11:1417-20
Kragol, G; Lovas, S; Varadi, G et al. (2001) The antibacterial peptide pyrrhocoricin inhibits the ATPase actions of DnaK and prevents chaperone-assisted protein folding. Biochemistry 40:3016-26
Otvos Jr, L (2000) Antibacterial peptides isolated from insects. J Pept Sci 6:497-511
Otvos Jr, L; Pease, A M; Bokonyi, K et al. (2000) In situ stimulation of a T helper cell hybridoma with a cellulose-bound peptide antigen. J Immunol Methods 233:95-105
Otvos Jr, L; O, I; Rogers, M E et al. (2000) Interaction between heat shock proteins and antimicrobial peptides. Biochemistry 39:14150-9
Otvos Jr, L; Bokonyi, K; Varga, I et al. (2000) Insect peptides with improved protease-resistance protect mice against bacterial infection. Protein Sci 9:742-9
McManus, A M; Otvos Jr, L; Hoffmann, R et al. (1999) Conformational studies by NMR of the antimicrobial peptide, drosocin, and its non-glycosylated derivative: effects of glycosylation on solution conformation. Biochemistry 38:705-14

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