ИСТИНА

Background: Pregnancy associated plasma protein-A (PAPP-A) dependent cleavage of IGF binding protein 4 (IGFBP-4) results in the release of active IGF and N- and C-terminal proteolytic fragments (NT- and CT-IGFBP-4). These fragments were recently shown to be strong biomarkers of adverse cardiac events risk in acute coronary syndrome (ACS) patients. Circulating IGFBP-4 is partially glycosylated in the N-terminal part. The impact of this glycosylation on IGFBP-4 proteolysis and the formation of NT- and CT-IGFBP-4 are still unknown. The aim of this study was to investigate the possible influence of glycosylation of IGFBP-4 on the formation of NT- and CT-IGFBP-4. Methods: Glycosylated and non-glycosylated IGFBP-4 and NT-IGFBP-4 were extracted from plasma samples of ACS patients (n=12) usinf immunochromatography and concanavalin A chromatography. The proteins were analyzed by mass-spectrometry (MS), western blotting (WB), and by using specific sandwich immunoassays. Precise molecular masses were obtained using mass-spectrometry (MS). IGFBP-4 glycan structure was investigated utilizing specific glycosidases. Results: The presence of glycosylated NT-IGFBP-4 (17260 Da) in concanavalin A purified preparation was confirmed by MS. The purified non-glycosylated IGFBP-4 was 3-4 times more susceptible to PAPP-A dependent proteolysis that the glycosylated IGFBP-4. The analysis of individual ACS plasma samples showed that 47.2-61.7% of full size IGFBP-4 was glycosylated, whereas only 9.8-23.5% of the NT-IGFBP-4 was glycosylated. Conclusion: The PAPP-A dependent proteolysis studies using purified glycosylated and non-glycosylated IGFBP-4 showed that the glycosylation of IGFBP-4 is able to suppress its specific proteolysis by PAPP-A. This is supported by the fact that the amount of glycosylated NT-IGFBP-4 detected in ACS plasma was lower than expected if both forms of IGFBP-4 were to be cleaved with similar efficiency.