A novel truncated form of apolipoprotein A-I transported by dense LDL is increased in diabetic patients

Abstract

Diabetic (DM) patients have exacerbated atherosclerosis and high CVD burden. Changes in lipid metabolism, lipoprotein structure, and dysfunctional HDL are characteristics of diabetes. Our aim was to investigate whether serum ApoA-I, the main protein in HDL, was biochemically modified in DM patients. By using proteomic technologies, we have identified a 26 kDa ApoA-I form in serum. MS analysis revealed this 26 kDa form as a novel truncated variant lacking amino acids 1-38, ApoA-IΔ(1-38). DM patients show a 2-fold increase in ApoA-IΔ(1-38) over nondiabetic individuals. ApoA-IΔ(1-38) is found in LDL, but not in VLDL or HDL, with an increase in LDL3 and LDL4 subfractions. To identify candidate mechanisms of ApoA-I truncation, we investigated potentially involved enzymes by in silico data mining, and tested the most probable molecule in an established animal model of diabetes. We have found increased hepatic cathepsin D activity as one of the potential proteases involved in ApoA-I truncation. Cathepsin D-cleaved ApoA-I exhibited increased LDL binding affinity and decreased antioxidant activity against LDL oxidation. In conclusion, we show for the first time: a) presence of a novel truncated ApoA-I form, ApoA-IΔ(1-38), in human serum; b) ApoA-IΔ(1-38) is transported by LDL; c) ApoA-IΔ(1-38) is increased in dense LDL fractions of DM patients; and d) cathepsin D-ApoA-I truncation may lead to ApoA-IΔ(1-38) binding to LDLs, increasing their susceptibility to oxidation and contributing to the high cardiovascular risk of DM patients.

Keywords: diabetes; hyperglycemia; low density lipoprotein; proteomics.

Fig. 1.

Representative serum 2-DE images (pI range 4–7, 10% PAGE) of ApoA-I spots in nonDM subjects (N = 6) (A) and DM-patients [N = 12 (6 type 1 and 6 type 2)] (B). C: Box-plot diagrams showing the intensity of each spot in both groups. Spot 5 was significantly increased in DM patients. **P < 0.01.

Fig. 2.

A: Representative serum 2-DE image (pI range 4–7, 10% PAGE) showing ApoA-I spot 6 at 26 kDa. B: Box-plot diagram showing the increase in spot 6 intensity in DM patients. **P < 0.01. C: MALDI-TOF spectra of human serum ApoA-I. D: Enlarged image showing the absence of the m/z 1,235.63 peak in spot 6 compared with spots 1–5.

Fig. 3.

Scheme showing the truncation of aas 1-38 in ApoA-I sequence and the fragments corresponding to the peaks detected in MALDI-TOF analysis. *Previously described ApoA-I forms (30) include: Pro-ApoA-I (aas 19-267) that corresponds to spots 4 and 5 in Fig. 1A, B, and mature ApoA-I (aas 25-267) that corresponds to spots 1, 2, and 3 in Fig. 1A, B. Spot 6 in Fig. 2A (identified here), corresponds to aas from 39 to 267, ApoA-IΔ(1-38).

Fig. 4.

A: Representative 2-DE image (pI range 4.7–5.9, 15% PAGE) of ApoA-I profile in serum, LPDS (N = 3 per group), HDL (DM, N = 6; nonDM, N = 9), and LDL (N = 5 per group) samples showing ApoA-IΔ(1-38) presence in serum and LDLs. B: Representative 1-DE and 2-DE Western blot (WB) images showing ApoA-I at 28 and 26 kDa in LDL samples. C: Box-plot diagram of the ApoA-IΔ(1-38) increase in LDLs of DM patients. *P < 0.05.

Fig. 5.

A: Representative 2-DE image (pI range 4–7, 10% PAGE) of ApoA-I in LDL subclasses in DM patients and nonDM subjects (30 DM patients and 30 nonDM individuals pooled in three groups of 10 subjects each). Bar diagrams showing the significant increase in ApoA-IΔ(1-38) intensity in LDL3 and LDL4 of DM patients (B) and in the sum of ApoA-IΔ(1-38) intensity in all the LDL subfractions (C). *P < 0.05.

Fig. 6.

Bar diagrams showing: the differential binding ability of full-length and cathepsin D-cleaved ApoA-I to HDLs and LDLs (***p<0.0001 for all comparisons; N = 3 independent experiments) (A); and the significant decrease in ApoA-I antioxidant capacity against LDL oxidation after cathepsin D truncation (***P < 0.0001 vs. LDL; §P < 0.0001 vs. full-length ApoA-I; †P < 0.0001 vs. truncated ApoA-I; N = 3 independent experiments) (B).

Fig. 7.

Scheme of the canonical pathway of the conversion of an ApoA-I molecule into mature HDL micelles showing the proposed pathway of the binding of ApoA-IΔ(1-38) to LDL particles.