ESI-MS quantitation of increased sphingomyelin in Niemann-Pick disease type B HDL

We have previously reported preferential release of polyunsaturated FAs during hydrolysis of lipoprotein phosphatidylcholine (PtdCho) by group X secretory phospholipase A 2 (sPLA 2 ) and preferential release of oligounsaturated FAs during hydrolysis of lipoprotein PtdCho by group V sPLA 2 , but the mechanism of this selectivity has remained unknown. We now show that the rate and specificity of hydrolysis are affected by relative increases in endogenous SM and free cholesterol (FC) during the lipase digestion. The highest preference for arachidonate release from LDL and HDL by group X sPLA 2 was observed for residual SM/PtdCho molar ratio of 1.2 and 0.4, compared with the respective starting ratios of 0.4 and 0.2, as measured by liquid chromatography/electrospray ionization-mass spectrometry. Group V sPLA 2 showed preferential release of linoleate from LDL and HDL at SM/PtdCho ratio 1.5 and 0.6, respectively. We have attributed the change in FA specificity to segregation of molecular species of PtdCho and of sPLA 2 s between disordered and ordered SM/FC/ PtdCho lipid phases. The increases in SM and FC during digestion with group IIA sPLA 2 were more limited, and a preferential hydrolysis of any FAs was not observed. A preferential release of linoleate during hydrolysis of phosphatidylcholine (PtdCho) by group V secretory phospholipase A 2 (sPLA 2 ) was first reported in liposomal (1-3) and later in lipoprotein (4-6) incubations, but the mechanism of this selectivity has remained unknown. Early work (1, 2) attributed the selective enzyme activity to a difference in the physical state of substrate between the sonicate and the natural membranes that may contain a more optimum mixture of phospholipids. Alternatively, a preferential release of arachidonate by group V sPLA 2 exogenously added to cell cultures has been attributed to its combined action with cytosolic PLA 2 (7). Subsequent work with liposomes suggested a special role for ceramides (3) in regulation of group V sPLA 2 hydrolysis of plasma PtdCho, which, however, was difficult to reconcile with the apparent absence of sphingomyelinase activity from plasma lipoproteins (6,8,9). Likewise, the apparent preferential release of PtdCho arachidonate by group X sPLA 2 during liposomal (10, 11), lipoprotein (4-6), and cellular (10, 12) incubations has remained unexplained. In contrast, group IIA sPLA 2 apparently attacked liposomal and lipoprotein PtdCho without FA discrimination, although at a significantly lower rate than group V and group X sPLA 2 s (3,5,9,10,12). Therefore, the true differences in the activity and apparent FA specificity of PtdCho hydrolysis by the sPLA 2 s have remained obscure.In other studies, a preferential release of arachidonate by group IIA sPLA 2 from phosphatidylethanolamine/ phosphatidylserine (PtdEtn/PtdSer) micelles was observed (13,14) in the presence of added SM/ceramide and was attributed to phase separation and exclusion of polyunsaturates from the ordered liquid phase formed under these conditions. The inhibitory effect...

Section: Apparent Absence Of Enzyme Specificity During Initial Digestionsupporting

confidence: 52%

Section: Role Of Cholesterolmentioning

confidence: 99%

Phase composition of lipoprotein SM/cholesterol/PtdCho affects FA specificity of sPLA2s

Kuksis

Pruzanski

2008

“…ApoA-I concentration in nascent LpA-I was determined by ELISA. Phospholipid concentrations in nascent LpA-I were determined by ESI-MS as we have previously described ( 20 ). 2D-PAGGE and non-denaturing (ND)-PAGGE were performed as described previously ( 18 ).…”

Section: Lipid and Lipoprotein Assaysmentioning

confidence: 99%

Analysis of lipid transfer activity between model nascent HDL particles and plasma lipoproteins: implications for current concepts of nascent HDL maturation and genesis

Bailey

Ruel

Hafiane

et al. 2010

Self Cite

The process of lipidation of apolipoprotein A-I (apoA-I) by the ABCA1 transporter is functionally important in the biogenesis of HDL and in regulating lipid transport and metabolism, which, in turn, is critical for normal human physiology. This process is believed to be one of the major mechanisms by which HDL may protect against atherosclerotic cardiovascular disease ( 1, 2 ). As a result, the molecular mechanisms underlying the origin of plasma HDL have been a subject of intense study.Although it is well established that the liver and intestine are major sources of newly secreted HDL ( 3, 4 ), there is little information on the metabolism of nascent HDL, their interaction with resident plasma lipoproteins, and their effect on lipid transport. Although discoidal nascent HDLs are believed to be critical intermediates between lipid-poor apoA-I and mature spherical HDL, the accurate detection and analysis of these nascent particles has proven diffi cult because they are rapidly remodeled by plasma factors and are subsequently found at relatively low concentrations in the plasma of most species. Abbreviations: 2D-PAGGE, two-dimensional polyacrylamide nondenaturing gradient gel electrophoresis; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fl uoride hydrochloride; apo, apolipoprotein; CE, cholesteryl ester; CETP, cholesteryl ester transfer protein; LpA-I, nascent apoA-I-containing particle; ND, non-denaturing; PC, phosphatidylcholine; PEG, polyethylene glycol; PL, phospholipid; PLTP, phospholipid transfer protein; r(HDL), reconstituted apoA-I-containing proteoliposome; TD, Tangier disease; UC, unesterifi ed cholesterol. Abstract

“…In this experiment, only the major SM and PC species were considered for the absolute quantitation, as we have reported previously (18). Compared with normal fibroblasts, LpA-I particles generated by incubation of lipid-free apoA-I with NPD-B fibroblasts had an average increase of z50% in SM levels ( Table 2).…”

Section: Npd-b Patients and Control Subjectsmentioning

confidence: 99%

“…The cellular protein concentration was measured using the Lowry method and was used to normalize the lipid quantitation. For PL mass analysis on HDL samples, electrospray ionization-mass spectrometry (ESI-MS) was used instead because of its higher sensitivity and unmatched accuracy for our highly purified analytes of limited quantity (18). ESI-MS analysis was carried out in positive and negative ion modes using a Micromass Quattro II triple quadrupole mass spectrometer equipped with an electrospray source, as we have reported previously (18){22}.…”

Section: Lipid Mass Quantitationmentioning

confidence: 99%

Increased sphingomyelin content impairs HDL biogenesis and maturation in human Niemann-Pick disease type B

Lee

Lesimple

Denis

et al. 2006

Self Cite

We previously reported that human Niemann-Pick Disease type B (NPD-B) is associated with low HDL. In this study, we investigated the pathophysiology of this HDL deficiency by examining both HDL samples from NPD-B patients and nascent high density lipoprotein (LpA-I) generated by incubation of lipid-free apolipoprotein A-I (apoA-I) with NPD-B fibroblasts. Interestingly, both LpA-I and HDL isolated from patient plasma had a significant increase in sphingomyelin (SM) mass (z50-100%). Analysis of LCAT kinetics parameters (V max and K m ) revealed that either LpA-I or plasma HDL from NPD-B, as well as reconstituted HDL enriched with SM, exhibited severely decreased LCAT-mediated cholesterol esterification. Importantly, we documented that SM enrichment of NPD-B LpA-I was not attributable to increased cellular mass transfer of SM or unesterified cholesterol to lipid-free apoA-I. Finally, we obtained evidence that the conditioned medium from HUVEC, THP-1, and normal fibroblasts, but not NPD-B fibroblasts, contained active secretory sphingomyelinase (S-SMase) that mediated the hydrolysis of [ 3 H]SM-labeled LpA-I and HDL 3 . Furthermore, expression of mutant SMase (DR608) in CHO cells revealed that DR608 was synthesized normally but had defective secretion and activity. Our data suggest that defective S-SMase in NPD leads to SM enrichment of HDL that impairs LCAT-mediated nascent HDL maturation and contributes to HDL deficiency. Thus, S-SMase and LCAT may act in concert and play a crucial role in the biogenesis and maturation of nascent HDL particles. Supplementary key words high density lipoprotein . nascent LpA-I . phospholipids . sphingomyelin phosphodiesterase-1 gene . sphingomyelinase Sphingomyelin (SM) plays an important role in the structural integrity of the cellular membranes. The constitutive degradation of SM occurs in the lysosomal compartments of the cell. Fragments of the plasma membrane containing SM targeted for degradation are endocytosed and traffic through the endosomal compartments to reach the lysosomes, where they are hydrolyzed by lysosomal sphingomyelinase. The cleaved fragments (i.e., the choline head group, the fatty acids, and the sphingoid bases) then leave the lysosome and reenter the biosynthetic pathway or can be further degraded. In Niemann-Pick type I disease, which includes types A and B (NPD-A/B), lysosomal SMase is deficient, resulting in a lysosomal accumulation of SM and a secondary increase in cholesterol (1).Earlier studies by Tall and colleagues (2) investigated the crucial role of phospholipids (PLs) and their plasma transfer proteins in the metabolic pathway of HDL. It is well accepted that SM plays an important role in plasma lipoprotein metabolism. SM is the second most abundant PL in mammalian plasma. It appears in all major lipoproteins, where it is part of the monolayer of polar lipids and cholesterol that surrounds a core of neutral lipids. Up to 18% of total plasma PL occurs as SM, and the ratio of SM-to-phosphatidylcholine (PC) varies widely among lipoprotein su...