出版社：American Society for Biochemistry and Molecular Biology
摘要：We previously reported that human Niemann-Pick Disease typeB (NPD-B) is associated with low HDL. In this study, we investigatedthe pathophysiology of this HDL deficiency by examining bothHDL samples from NPD-B patients and nascent high density lipoprotein(LpA-I) generated by incubation of lipid-free apolipoproteinA-I (apoA-I) with NPD-B fibroblasts. Interestingly, both LpA-Iand HDL isolated from patient plasma had a significant increasein sphingomyelin (SM) mass (50–100%). Analysis of LCATkinetics parameters (Vmax and Km) revealed that either LpA-Ior plasma HDL from NPD-B, as well as reconstituted HDL enrichedwith SM, exhibited severely decreased LCAT-mediated cholesterolesterification. Importantly, we documented that SM enrichmentof NPD-B LpA-I was not attributable to increased cellular masstransfer of SM or unesterified cholesterol to lipid-free apoA-I.Finally, we obtained evidence that the conditioned medium fromHUVEC, THP-1, and normal fibroblasts, but not NPD-B fibroblasts,contained active secretory sphingomyelinase (S-SMase) that mediatedthe hydrolysis of [3H]SM-labeled LpA-I and HDL3. Furthermore,expression of mutant SMase (R608) in CHO cells revealed thatR608 was synthesized normally but had defective secretion andactivity. Our data suggest that defective S-SMase in NPD leadsto SM enrichment of HDL that impairs LCAT-mediated nascent HDLmaturation and contributes to HDL deficiency. Thus, S-SMaseand LCAT may act in concert and play a crucial role in the biogenesisand maturation of nascent HDL particles.Supplementary key words high density lipoprotein • nascent LpA-I • phospholipids • sphingomyelin phosphodiesterase-1 gene • sphingomyelinase
Abbreviations: apoA-I, apolipoprotein A-I; CE, cholesteryl ester; ESI-MS, electrospray ionization-mass spectrometry; FC, free cholesterol; HDL-C, high density lipoprotein-cholesterol; LpA-I, nascent apoA-I-containing particle; NPD-A/B, Niemann-Pick disease type A and B; PC, phosphatidylcholine; PL, phospholipid; rLpA-I, reconstituted apolipoprotein A-I-containing proteoliposomes; SM, sphingomyelin; SMase, sphingomyelinase; SMPD-1, sphingomyelin phosphodiesterase-1 gene; S-SMase, secretory sphingomyelinase; SR-BI, scavenger receptor class B type I; TD, Tangier disease; 2D-PAGGE, two-dimensional polyacrylamide nondenaturing gradient gel electrophoresis; 22OH/9CRA, 2.5 µg/ml 22(R)-hydroxycholesterol and 5 µM 9-cis-retinoic acid
50–100%). Analysis of LCAT kinetics parameters (Vmax and Km) 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 [3H]SM-labeled LpA-I and HDL3. Furthermore, expression of mutant SMase (
R608) in CHO cells revealed that 
