Phospholipid transfer protein (PLTP) causes proteolytic cleavage of apolipoprotein A-I

Abstract

Plasma phospholipid transfer protein (PLTP) is a factor that plays an important role in HDL metabolism. In this study we present data suggesting that PLTP has an inherent protease activity. After incubation of HDL 3 in the presence of purified plasma PLTP, the d Ͻ 1.25 g/ml particles (fusion particles) contained intact 28.2 kDa apoA-I while the d Ͼ 1.25 g/ml fraction (apoA-I-PL complexes) contained, in addition to intact apoA-I, a cleaved 23 kDa form of apoA-I. Purified apoA-I was also cleaved by PLTP and produced a similar 23 kDa apoA-I fragment. The cleavage of apoA-I increased as a function of incubation time and the amount of PLTP added. The process displayed typically an 8-10 h lag or induction period, after which the cleavage proceeded in a time-dependent manner. This lagphase was necessary for the development of the cleavage activity during incubation at 37 ؇ C. The specific apoA-I cleavage activity of different PLTP preparations varied between 0.4-0.8 g apoA-I degraded/h per 1000 nmol per h of PLTP activity. The 23 kDa apoA-I fragment reacted with monoclonal antibodies specific for the N-terminal part of apoA-I, indicating that the apoA-I cleavage occurred in the Cterminal portion. The apoA-I cleavage products were further characterized by mass spectrometry. The 23 kDa fragment yielded a mass of 22.924 kDa, demonstrating that the cleavage occurs in the C-terminal portion of apoA-I between amino acid residues 196 (alanine) and 197 (threonine). The intact apoA-I and the 23 kDa fragment revealed identical Nterminal amino acid sequences. The cleavage of apoA-I could be inhibited with APMSF and chymostatin, suggesting that it is due to a serine esterase-type of proteolytic activity. Recombinant PLTP produced in CHO cells or using the baculovirus-insect cell system caused an apoA-I cleavage pattern identical to that obtained with plasma PLTP. The present results raise the question of whether PLTP-mediated proteolytic cleavage of apoA-I might affect plasma HDL metabolism by generating a novel kinetic compartment of apoA-I with an increased turnover rate.

Distribution of phospholipid transfer protein in human plasma: presence of two forms of phospholipid transfer protein, one catalytically active and the other inactive

“…Recombinant human PLTP (rhPLTP) was prepared in Chinese hamster ovary (CHO) cells and purified as described (23). Human plasma PLTP was purified as described (15).…”

ProteinChip Technology: A New and Facile Method for the Identification and Measurement of High-Density Lipoproteins apoA-I and apoA-II and Their Glycosylated Products in Patients with Diabetes and Cardiovascular Disease

“…However, due to significant cross-reactivity between specific antibodies used in these studies, the immunoassays do not accurately reflect the level of apolipoproteins in the plasma. [18][19][20][21][22][23][24][25][26][27][28][29] Also, accurate measurement of both apolipoprotein isoforms could be of metabolic interest since the two major HDL apolipoproteins have been shown to differentially modulate cholesteryl ester transfer protein (CETP) activity and phospholipid transfer protein (PLTP) activity. [20][21][22][23][24][25][26][27][28][29][30][31] It is now well established that plasma lipoproteins do not constitute stable entities in vivo but rather are continuously modified in the blood stream through the action of various enzymes, lecithin:cholesterol acyltransferase (LCAT), lipoprotein lipase (LP), cholesteryl ester transfer protein (CETP), and the phospholipid transfer protein (PLTP).…”

“…[18][19][20][21][22][23][24][25][26][27][28][29] Also, accurate measurement of both apolipoprotein isoforms could be of metabolic interest since the two major HDL apolipoproteins have been shown to differentially modulate cholesteryl ester transfer protein (CETP) activity and phospholipid transfer protein (PLTP) activity. [20][21][22][23][24][25][26][27][28][29][30][31] It is now well established that plasma lipoproteins do not constitute stable entities in vivo but rather are continuously modified in the blood stream through the action of various enzymes, lecithin:cholesterol acyltransferase (LCAT), lipoprotein lipase (LP), cholesteryl ester transfer protein (CETP), and the phospholipid transfer protein (PLTP). 4,[20][21][22][23][24][25][26][27][28][29][30][31] Recent studies have demonstrated that the two distinct plasma lipid transfer proteins, CETP and PLTP, markedly alter the size distribution of plasma lipoprotein fractions by shuttling lipid components from one lipoprotein substrate to another.…”

Phosphorylcholine-Installed Nanocarriers Target Pancreatic Cancer Cells through the Phospholipid Transfer Protein

“…Next, to examine the role of PLTP on the cellular uptake of PC-8-PEG, we inhibited PLTP with thimerosal at 0.1 mM. 30 The inhibition of PLTP considerably suppressed the cellular uptake of PC-8-PEG (Figure 2B,C), supporting the role of PLTP as a target of PC-8-PEG and a mediator of the enhanced cellular uptake.…”