Proteolytic cleavage sites

The fluorescent beads are manually isolated, washed with 8 M guanidine-HCl and water, and then submitted for sequence analysis. We expect that some of the peptides on the positive beads will be cleaved at the proteolytic site. As a result, the PTH-amino acids detected during each cycle of Edman degradation will have been generated from both of these peptides. This complicates the sequence analysis somewhat. However, it also allows us to determine the proteolytic cleavage site of the peptide, in addition to its uncleaved sequence. 

Figure 1. Diagram of the human thrombin receptor, viewed edge-on, showing the sequence around the proteolytic cleavage site (LDPR-SFLLRN) in the long extracellular amino-terminus and the seven transmembrane domains (TM1-TM7).

Figure 2. Schematic representation of pre-pro-Bombyx PTTH cDNA. Coding regions are indicated by boxes numerals in parenteses are the number of amino acid residues in the respective components. Putative proteolytic cleavage sites are indicated by KRK, KR, and RKR. Triangles represent the polyadenylated tract.

Figure 2. Diagram of the double loop and negatively charged domain of GPIba showing the mutations leading to platelet-type von Willebrand s disease, Gly233Val and Met239Val, as well as the major proteolytic cleavage sites.

Fig. 5. Proteolytic cleavage sites in apolipoprotein E. Top Linear representation of the structure of human apoE demonstrating cleavage sites when apoE3 was subjected to limited proteolysis with six enzymes O, trypsin 9, elastase , chymotrypsin O, subtili-sin , Staphylococcus aureus V8 , thrombin. Bottom Linear representation of the two thrombolytic fragments of apoE, the 22- and 10-kDa fragments.

SCF is a ligand that is expressed on Sertoli cells. A proteolytic cleavage site within exon 6 results in two isoforms of SCF, a soluble isoform (sSCF) and a membrane-bound isoform (mSCF). c-Kit, the SCF receptor, is a 145-kD transmembrane tyrosine kinase expressed on germ cells and Leydig cells, the... 

Asp mentioned above and Lys Ser, which was introduced in order to remove a proteolytic cleavage site [39]. Thus, there were altogether four fixed amino acid replacements in addition to fhe randomized side chains. The mutagenized gene cassette was fhen inserted into an appropriate E. coli vector and a genetic library comprising 3.7x10 variants was prepared [40]. 

Mullberg, J., Oberthur, W, Lottspeich, F., Mehl, E. Dittrich, E., Graeve, L., Heinrich, P C., and Rose-John, S. (1994). The soluble human IL-6 receptor. Mutational characterization of the proteolytic cleavage site. J Immunol. 752 4958-4968. 

Fig. 5. The principle of processing-independent immunoassays. The scheme illustrates the processing from translation of pre-pro-protein in the endoplasmatic reticulum, via intermediates in the Golgi apparatus and early secretory vesicles, to final maturation (see also Fig. 3). Divisions within the protein bars indicate proteolytic cleavage sites. The protuberances on the tops and ends of the bars illustrate different forms of amino acid derivatization. The filled-in areas of the protein bars are the selected processing-independent sequences, toward which the processing-independent immunoassay is developed.

PE is a single-chain protein with a molecular mass of 66 kDa composed of three distinct domains (Fig. 2). In the PE protein, domain I (1-252) binds to the PE receptor on normal animal cells, which has been identified as the a2-macroglobulin receptor. Domain II (253-364) mediates translocation of domain III (400-613) into the cytosol. The translocation domain contains a proteolytic cleavage site within a disulfide loop, which, after proteolytic cleavage, leaves the cell-binding site (I) and translocation domain (II) bound to the catalytic/toxic site (III) by a disulfide bond (Fig. 2). Following reduction of this bond in the cytosol, the ADP-ribosylation activity of domain III inactivates elongation factor (EF2) and causes inhibition of protein synthesis and cell death. 

Identify proteolytic cleavage sites in the peptide and design a strategy for fragment generation. 

Sokic, S., Papavasiliou, G., 2012. Controlled proteolytic cleavage site presentation in bio-mimetic PEGDA hydrogels enhances neovascularization in vitro. Tissue Eng. Part A 18,... 

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