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Cyst-13 complex. Literature data suggest that an epitope is generally composed of five toseven core amino acids and up to 10 contact residues. Our results correspond to these data,as we identified eight amino acid residues as the direct epitope, but in an affinityexperiment with the synthetic peptide epitope, the octapeptide was not binding to theantibody. Prolongation of the epitope from the N-terminus by two residues resulted in thedecapeptide PWQGTMTLSK showing much higher binding ability in affinity experiments.It was proved that prolongation of the epitope sequence by two residues stabilizes thecomplex and ensures the specificity of the molecular recognition. The determined epitopesequence is located in the C-terminal region of cystatin C which is exposed to theenvironment, therefore there is no steric hindrance which can impede interactions betweenhCC and the antibody. The epitope sequence (hCC 107-114) is located within the L2- 5strand (Figure 1).The identification of the binding site may be of high importance fordimerization/oligomerization and fibrillization studies of hCC. Gel electrophoresis provedthe direct influence of mAb Cyst-13 on the dimerization of hCC. The incubation of hCCwith mAb Cyst-13 showed the suppression of the dimerization process for all mAb/hCCmolar ratios applied. Even the lowest amount of the antibody slightly suppressed the dimerformation in comparison with the dimerization of the native protein (Figure 2). It is offurther interest to investigate how blocking of the C-terminal part by interaction with theantibody can influence the dimerization process.The production of specific antibodies in the human organism is an importantregulatory mechanism for different processes. Thanks to the use of the epitope extractionandexcision-mass spectrometry method we were able to identify successfully the epitopefor the monoclonal antibody Cyst-13 raised against human cystatin C. The epitopeidentification offer a possibility to use a more defined active immunization with a partialsequence of hCC conjugated to a carrier protein. This approach may yield a strong antibodyresponse to a definite region of cystatin C and is therefore promising in combatingpathological conditions related to hCC amyloidogenity (HCCAA).AcknowledgmentsThe work was supported by Ministry of Science and Higher Education, Grant No. 1264/H03/2009/37.References1. Nilsson, M.X. Wang, Rodziewicz-Motowidło, S., Jankowski, R., Lindstrom, V., Onnerfjord, P.,Westermark, G., Grzonka, Z., Jaskólski, M., Grubb, A. J. Biol. Chem. 279(23), 24236-45 (2004).2. Stefanescu, R., Iacob, R.E., Damoc, E.N., Marquardt, A., Amstalden, E., et al. Eur. J. Mass.Spectrom. (Chichester, Eng.) 13, 69-75 (2007).287