ИСТИНА

Background: N- and C-terminal proteolytic fragments of IGFBP-4 (NT-IGFBP-4, 14.6 kDa, and CT-IGFBP-4, 11.3 kDa) are strong predictors of major adverse cardiac events risk in patients presented with ischemia and stable patients with type 1 diabetes. The presence of these fragments in the circulation depends on the proteolytic activity of metalloprotease PAPP-A, which specifically cleaves IGFBP-4 between Met135 and Lys136. We have previously developed sandwich immunoassays based on monoclonal antibodies that are specific to proteolytic neo-epitopes on NT-IGFBP-4 and CT-IGFBP-4 and have less than 1% of cross-reactivity to the full-length IGFBP-4. It has been shown that a fraction of circulating IGFBP-4 contains glycosylated Asn104 that is located close to the PAPP-A-specific cleavage site. Our assay for NT-IGFBP-4 relies on an antibody that has a binding epitope which is in the vicinity of Asn104 and hence the NT-IGFBP-4 assay may be affected by the glycosylation of this amino acid residue. Therefore, the aims of this study were: a) To determine NT-IGFBP-4 glycosylation levels in individual EDTA-plasma samples and... b) To evaluate the influence of glycosylation on the immunodetection of NT-IGFBP-4. Methods: IGFBP-4 and its proteolytic fragments were extracted from twelve individual EDTA plasma samples of ACS patients by immunoprecipitation and analyzed using enhanced chemiluminescence immunoblotting (ECL). Concanavalin A sepharose was used to verify the presence of glycosylated forms of IGFBP-4 and NT-IGFBP-4. The levels of IGFBP-4 and NT-IGFBP-4 were measured using sandwich HRP immunoassays. The precise masses of purified glycosylated and nonglycosylated NT-IGFBP-4 were confirmed by mass spectrometry. Increases in the concentrations of the proteolytic fragments of IGFBP-4 during the incubation with recombinant PAPP-A were used as measures of the proteolysis rates of glycosylated and non-glycosylated IGFBP-4. Results: The investigation of individual EDTA plasma samples revealed that of the total circulating IGFBP-4, 47.2-61.7% was glycosylated. Meanwhile, of the total NT-IGFBP-4, only 9.8-23.5% was glycosylated. Mass spectrometric analysis of NTIGFBP- 4 extracted from pooled EDTA plasma revealed two peaks of 17260 and 14615 Da that corresponded to glycosylated and non-glycosylated forms respectively. The immunoreactivities of endogenous glycosylated and non-glycosylated NT-IGFBP-4 differed by less than 10% when measured using HRP or ECL based immunoassays. PAPP-A-dependent proteolysis of glycosylated IGFBP-4 was 3-4 times less efficient compared to proteolysis of non-glycosylated IGFBP-4. Conclusion: For the first time, the presence of glycosylated NT-IGFBP-4 in human plasma was shown and the proportion of glycosylated and non-glycosylated NTIGFBP- 4 was measured. PAPP-A-dependent proteolysis of glycosylated IGFBP-4 is less efficient if compared with the proteolysis of non-glycosylated IGFBP-4, although it is not completely inhibited. The glycosylated NT-IGFBP-4 displays the same immunoreactivity as non-glycosylated NT-IGFBP-4 in the fragment-specific immunoassay. Thus, this sandwich immunoassay can be used for the reliable measurement of NT-IGFBP-4 in the blood of patients.