Isolation and lipid characterization of cholesterol-enriched fractions in cortical and nuclear human lens fibers.
OBJECTIVE
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.
Rujoi
Madalina
M
Department of Chemistry, University of Louisville, Louisville, Kentucky 40292, USA.
Jin
Jiaoling
J
Borchman
Douglas
D
Tang
Daxin
D
Yappert
M Cecilia
MC
D000368:Aged; D000369:Aged, 80 and over; D015153:Blotting, Western; D002386:Cataract / Q000378:metabolism*; D021941:Caveolae / Q000737:chemistry; D002784:Cholesterol / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007904:Lens Cortex, Crystalline / Q000737:chemistry*; D007907:Lens Nucleus, Crystalline / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D021962:Membrane Microdomains / Q000737:chemistry; D010743:Phospholipids / Q000032:analysis* / Q000302:isolation & purification*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D010743:Phospholipids; D002784:Cholesterol