Structural and functional domains of apolipoprotein A-I within high density lipoproteins.

When stimulated, rat serosal mast cells degranulate and secrete a cytoplasmic neutral protease, chymase. We studied the fragmentation of apolipoprotein (apo) A-I during proteolysis of HDL 3 by chymase, and examined how chymase-dependent proteolysis interfered with the binding of eight murine monoclonal antibodies (Mabs) against functional domains of apoA-I. Size exclusion chromatography of HDL 3 revealed that proteolysis for up to 24 h did not alter the integrity of the ␣ -migrating HDL, whereas a minor peak containing particles of smaller size with pre ␤ mobility disappeared after as little as 15 min of incubation. At the same time, generation of a large (26 kDa) polypeptide containing the N-terminus of apoA-I was detected. This large fragment and other medium-sized fragments of apoA-I produced after prolonged treatment with chymase were found to be associated with the ␣ HDL; meanwhile, small lipid-free peptides were rapidly produced. Incubation of HDL 3 with chymase inhibited binding of Mab A-I-9 (specific for pre ␤ 1 HDL) most rapidly (within 15 min) of the eight studied Mabs. This rapid loss of binding was paralleled by a similar reduction in the ability of HDL 3 to induce high-affinity efflux of cholesterol from macrophage foam cells, indicating that proteolysis had destroyed an epitope that is critical for this function. In sharp contrast, prolonged degradation of HDL 3 by chymase failed to reduce the ability of HDL 3 to activate LCAT, even though it led to modification of three epitopes in the central region of apoA-I that are involved in lecithin cholesterol acyltransferase (LCAT) activation. This differential sensitivity of the two key functions of HDL 3 to the proteolytic action of mast cell chymase is compatible with the notion that, in reverse cholesterol transport, intactness of apoA-I is essential for pre ␤ 1 HDL to promote the high-affinity efflux of cellular cholesterol, but not for the ␣ -migrating HDL particles to activate LCAT. -Lee, M., P. Uboldi, D. Giudice, A. L. Catapano, and P. T. Kovanen. Identification of domains in apoA-I susceptible to proteolysis by mast cell chymase: implications for HDL function. J. Lipid Res. 41: 975-984.Supplementary key words ␣ -and pre ␤ -migrating HDL • monoclonal antibodies • LCAT • cellular cholesterol efflux • proteolysis of apoA-I

Section: Discussionsupporting

confidence: 93%

mentioning

confidence: 99%

Identification of domains in apoA-I susceptible to proteolysis by mast cell chymase: implications for HDL function

Lee

Uboldi

Giudice

et al. 2000

“…Whereas these proteases have provided a wealth of information on the structure of apoA-I, they do not lend themselves well to an assay designed to distinguish lipid-free and lipid-bound apoA-I. Chymotrypsin (42,43), V8 protease (41,43), trypsin (39)(40)(41)(42), elastase (41,42), and subtillisin (42) all rapidly digest lipid-free apoA-I into a complex set of peptide products. However, they also are capable of digesting lipid-bound apoA-I into a separate complex set of peptides, albeit at a significantly slower rate.…”

Section: The Benefits Of the Enteropeptidase Assay Versus Existing Approachesmentioning

confidence: 99%

A proteolytic method for distinguishing between lipid-free and lipid-bound apolipoprotein A-I

Safi

Maiorano

Davidson

2001

Recent studies indicate that certain lipid-poor forms of apolipoprotein (apo)A-I may be particularly important in promoting cholesterol release from overburdened cells in the periphery. However, a detailed understanding of the physiological relevance of these species has been hampered by the difficulty in measuring them. As part of a search for a rapid assay for these forms of apoA-I, we have observed that the protease enteropeptidase can specifically cleave human lipid-free apoA-I but not its lipid-bound form. Enteropeptidase cleaved lipid-free apoA-I at a single site at amino acid 188, resulting in an N-terminal fragment of 22 kDa. However, apoA-I was not susceptible to enteropeptidase when present in reconstituted high-density lipoprotein (rHDL) particles as small as 6 nm in diameter or in human HDL 3 particles, even at extremely high enzyme-to-protein ratios and extended reaction times. We capitalized on this observation to develop an assay for the measurement of lipidpoor apoA-I in in vitro systems. Densitometry was used to generate a standard curve from sodium dodecyl sulfate polyacrylamide gels to determine theamounts of the N-terminal proteolytic fragment in unknown samples treated with enteropeptidase. This system could accurately quantify apoA-I that had been displaced from rHDL particles and human HDL 3 with purified apoA-II. On the basis of the results, a system of nomenclature is proposed for "lipid-free," "lipid-poor," and "lipid bound" apoA-I. The reported method distinguishes forms of apoA-I by a conformational parameter without previous separation of the species. This simple and inexpensive method will be useful for understanding the characteristics of plasma HDL that are favorable for the dissociation of apoA-I.-Safi, W., J. N. Maiorano, and W. S. Davidson. A proteolytic method for distinguishing between lipid-free and lipid-bound apolipoprotein A-I. J. Lipid Res. 2001. 42: 864-872. Supplementary key words enteropeptidase • lipid-poor apoA-I • pre-␤ HDL • lipid-free apoA-I • lipoprotein • cholesterol methods This is an Open Access article under the CC BY license.

“…Only about 10% of the total particle mass is lipid. It has been suggested that the conformational state of apoA-I in pre ␤ -HDL exposes protease-sensitive regions, leading to enhanced predisposition to proteolytic cleavage (14,16,17). We report in this study that PLTP, in addition to being able to transfer phospholipids and release lipid-poor apoA-I from HDL during the conversion process, also is able to cause proteolytic cleavage of the main apolipoprotein of HDL, apoA-I.…”

Section: Phospholipid Transfer Protein (Pltp) Causes Proteolytic Cleavage Of Apolipoprotein A-imentioning

confidence: 68%

“…Proteolysis is known to regulate the clearance of several plasma proteins (49)(50)(51). Although the N-terminal domain of apoA-I can undergo limited proteolysis (52), the carboxy-terminal domain seems to be particularly sensitive to proteolysis as substantiated by previous reports where limited enzymatic proteolysis produced 22-26 kDa N-terminal fragments (16,17,41). These in vitro proteolytic studies suggest that enzymatic cleavage of apoA-I may also occur in vivo.…”

Section: Discussionmentioning

confidence: 87%

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

Jauhiainen

Huuskonen

Baumann

et al. 1999

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.