Abstract
Apolipoprotein A-Ib (ApoA-Ib) is a high molecular weight form of Apolipoprotein A-I (ApoA-I) found specifically in the urine of kidney-transplanted patients with recurrent idiopathic focal segmental glomerulosclerosis (FSGS). To determine the nature of the modification present in ApoA-Ib, we sequenced the whole APOA1 gene in ApoA-Ib positive and negative patients, and we also studied the protein primary structure using mass spectrometry. No genetic variations in the APOA1 gene were found in the ApoA-Ib positive patients that could explain the increase in its molecular mass. The mass spectrometry analysis revealed three extra amino acids at the N-Terminal end of ApoA-Ib that were not present in the standard plasmatic form of ApoA-I. These amino acids corresponded to half of the propeptide sequence of the immature form of ApoA-I (proApoA-I) indicating that ApoA-Ib is a misprocessed form of proApoA-I. The description of ApoA-Ib could be relevant not only because it can allow the automated analysis of this biomarker in the clinical practice but also because it has the potential to shed light into the molecular mechanisms that cause idiopathic FSGS, which is currently unknown.
Highlights
Idiopathic focal segmental glomerulosclerosis (FSGS) is a glomerular disease presumably caused by a still unknown circulating factor that affects the podocyte function by disrupting the glomerular filtration barrier, which causes heavy proteinuria[1,2,3,4]
Once assessed that the increase in molecular mass of Apolipoprotein A-Ib (ApoA-Ib) had not a genetic origin we evaluated the presence of possible post-translational modifications (PTMs) that could explain the increase in molecular weight, focusing in the presence of N or O-linked glycosylations on ApoA-Ib
Urinary ApoA-Ib, a high molecular weight form of Apolipoprotein A-I (ApoA-I) discovered by our group, has proven to be related to idiopathic FSGS recurrence after kidney transplantation[7,8]
Summary
Idiopathic focal segmental glomerulosclerosis (FSGS) is a glomerular disease presumably caused by a still unknown circulating factor that affects the podocyte function by disrupting the glomerular filtration barrier, which causes heavy proteinuria[1,2,3,4]. Plasmatic ApoA-I forms appear as a train of five spots in a two dimension SDS-PAGE gels, where the central is the most abundant and named ApoA-I form 015,16 Taking this form as a reference, other mature ApoA-I forms with similar molecular mass but more acidic isoelectric point (IEP) probably caused by post translational modifications[17] correspond to spots −2, −1. Forms of ApoA-I around 50 kDa have been detected in blood related to cardiovascular disease events[24,25] and in urine of pediatric FSGS patients[9] The origin of this 50 kDa variants is unclear but its molecular mass and electrophoretic properties certainly differ from ApoA-Ib9,26,27. The knowledge of the molecular primary structure of ApoA-Ib could allow the design of either specific antibodies against ApoA-Ib or targeted proteomic methods allowing automated ApoA-Ib analyses and quantification in the clinical laboratory setting for diagnostic purposes
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