人晶状体皮质和核中富含胆固醇组分的分离及脂质特性分析

Isolation and lipid characterization of cholesterol-enriched fractions in cortical and nuclear human lens fibers.

作者信息

Rujoi Madalina, Jin Jiaoling, Borchman Douglas, Tang Daxin, Yappert M Cecilia

机构信息

Department of Chemistry, University of Louisville, Louisville, Kentucky 40292, USA.

出版信息

Invest Ophthalmol Vis Sci. 2003 Apr;44(4):1634-42. doi: 10.1167/iovs.02-0786.

DOI:10.1167/iovs.02-0786

PMID:12657603

Abstract

PURPOSE

Human lens membranes contain unusually high levels of cholesterol and sphingolipids, lipids known to segregate into liquid-ordered domains. The current study was conducted to pursue the determination and characterization of these domains in membranes of clear and cataractous human lenses.

METHODS

Cortical and nuclear regions of aged clear and cataractous lenses were obtained. After lysis with Triton X-100 at 4 degrees C and sucrose linear-density centrifugation, sedimenting and nonsedimenting fractions (when present) were collected. Phospholipids were analyzed by (31)P-nuclear magnetic resonance (NMR) and mass spectrometry. Caveolae and raft markers were tested by Western blot analysis.

RESULTS

Only samples from clear lenses exhibited a nonsedimenting band. Phospholipid contents were comparable for sedimenting fractions of clear and cataractous membranes. Cholesterol to phospholipid molar ratios in light-density bands were nearly 7, three times greater than in sedimenting fractions. The portion of total cholesterol present in nonsedimenting fractions increased from 5.5% in the cortex to 14% in the nucleus. Two lysophospholipids comprising approximately 10% of all phospholipids in total membranes were undetectable in nonsedimenting fractions. Caveolin-1 was enriched in these fractions.

CONCLUSIONS

Phospholipid compositional differences between lighter and heavier fractions from clear lenses were relatively minor and could not, alone, account for the substantial enrichment of cholesterol in the lighter fractions. Specific proteins, such as caveolin-1, must recruit cholesterol and induce clustering. Undetectable amounts of light-density domains in cataractous membranes suggest either disruption of these aggregates and thus the function of proteins within them, possibly relevant to lens transparency, and/or greater density of these clusters due to stronger binding of insoluble crystallins to membranes.

摘要

目的

人晶状体膜含有异常高水平的胆固醇和鞘脂，这些脂质已知会分离成液晶相结构域。本研究旨在确定和表征透明和白内障人晶状体膜中的这些结构域。

方法

获取老年透明和白内障晶状体的皮质和核区域。在4℃用 Triton X - 100裂解并经蔗糖线性密度离心后，收集沉淀和非沉淀级分（如果存在）。通过（31）P - 核磁共振（NMR）和质谱分析磷脂。通过蛋白质免疫印迹分析检测小窝和脂筏标志物。

结果

仅透明晶状体的样品显示出非沉淀带。透明和白内障膜沉淀级分中的磷脂含量相当。轻密度带中胆固醇与磷脂的摩尔比接近7，是沉淀级分中的三倍。非沉淀级分中总胆固醇的比例从皮质中的5.5％增加到核中的14％。在非沉淀级分中未检测到占总膜中所有磷脂约10％的两种溶血磷脂。小窝蛋白 - 1在这些级分中富集。

结论

透明晶状体较轻和较重级分之间的磷脂组成差异相对较小，单独无法解释较轻级分中胆固醇的大量富集。特定蛋白质，如小窝蛋白 - 1，必须募集胆固醇并诱导聚集。白内障膜中未检测到轻密度结构域，这表明这些聚集体可能被破坏，从而破坏其中蛋白质的功能，这可能与晶状体透明度有关，和/或由于不溶性晶状体蛋白与膜的更强结合导致这些聚集体的密度更高。