Characterization of the Purified RON and RON in the Crystals
The purified RON Sema-PSI-IPT1 has a molecular mass of 77,114±91 Da, consistent with the five predicted glycosylation sites. The electron density map, however, accounted only for the Sema and PSI domains. SDS-PAGE analysis of the crystals suggested degradation of the protein during crystallization. The mass spectrometry analysis of RON crystals revealed a major peak of 65,616±235 Da. Crystal packing positions Val42 as the first residue seen in the electron density map. Val42 is located in a highly crowded environment with no space to accommodate the preceding amino acids. The lower than expected molecular weight and the arrangement in the crystal suggest a proteolytic cleavage of the 17 N-terminal residues. Moreover, western analysis of RON crystals showed a truncated protein that was not recognized by the C-terminal specific Penta-His antibody, indicating additional loss of C-terminal residues. Thus, a second proteolytic cleavage site occurred in the IPT1 domain. Accounting for the loss of the 17 N-terminal residues, the difference between the calculated and experimentally determined molecular mass implies the degradation of approximately 75 of the 115 IPT1 residues. No electron density was associated with these remaining IPT1 residues and accordingly, they were not modeled. The Drosophila S2 cells produce at least four acid active cathepsins [60], and trace amounts of contaminating proteases in the RON Sema-PSI-IPT1 preparation could cleave the protein at the low pH (4.6) of the crystal growth solution. In contrast, the RON Sema-PSI-IPT1 protein remained intact when stored at pH 8.0. However, the loss of the C-terminal residues of IPT1 during crystallization may be related to the trypsin-sensitive Lys632-Lys633 peptide bond of IPT1 [49]. Ma and colleagues concluded that the susceptibility of RON IPT1 to the cell-associated trypsin-like proteases regulates RON mediated tumorigenic activities in epithelial cells.