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J. Biol. Chem., Vol. 281, Issue 41, 30660-30668, October 13, 2006

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Caveolar Endocytosis and Microdomain Association of a Glycosphingolipid Analog Is Dependent on Its Sphingosine Stereochemistry^*

Raman Deep Singh, Yidong Liu, Christine L. Wheatley, Eileen L. Holicky, Asami Makino^¶, David L. Marks, Toshihide Kobayashi^¶, Gopal Subramaniam, Robert Bittman, and Richard E. Pagano¹

From the Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905, the Department of Chemistry and Biochemistry, Queens College, The City University of New York, Flushing, New York 11367, and the ^¶RIKEN Frontier Research System, Wako, Saitama 351-0198, Japan

We have previously shown that glycosphingolipid analogs are internalized primarily via caveolae in various cell types. This selective internalization was not dependent on particular carbohydrate headgroups or sphingosine chain length. Here, we examine the role of sphingosine structure in the endocytosis of BODIPY^TM-tagged lactosylceramide (LacCer) analogs via caveolae. We found that whereas the LacCer analog with the natural (D-erythro) sphingosine stereochemistry is internalized mainly via caveolae, the non-natural (L-threo) LacCer analog is taken up via clathrin-, RhoA-, and Cdc42-dependent mechanisms and largely excluded from uptake via caveolae. Unlike the D-erythro-LacCer analog, the L-threo analog did not cluster in membrane microdomains when added at higher concentrations (5–20 µM). In vitro studies using small unilamellar vesicles and giant unilamellar vesicles demonstrated that L-threo-LacCer did not undergo a concentration-dependent excimer shift in fluorescence emission such as that seen with BODIPY^TM-sphingolipids with natural stereochemistry. Molecular modeling studies suggest that in D-erythro-LacCer, the disaccharide moiety extends above and in the same plane as the sphingosine hydrocarbon chain, while in L-threo-LacCer the carbohydrate group is nearly perpendicular to the hydrocarbon chain. Together, these results suggest that the altered stereochemistry of the sphingosine group in L-threo-LacCer results in a perturbed structure, which is unable to pack closely with natural membrane lipids, leading to a reduced inclusion in plasma membrane microdomains and decreased uptake by caveolar endocytosis. These findings demonstrate the importance of the sphingolipid stereochemistry in the formation of membrane microdomains.

Received for publication, June 28, 2006 , and in revised form, July 31, 2006.

^* This work was supported by National Institutes of Health Grants GM-22942 (to R. E. P.) and HL-083187 (to R. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1–3 and two movies.

¹ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, 200 First St. SW, Rochester, MN 55905. Tel.: 507-284-8754; Fax: 507-266-4413; E-mail: pagano.richard{at}mayo.edu.

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				R. D. Singh, E. L. Holicky, Z.-j. Cheng, S.-Y. Kim, C. L. Wheatley, D. L. Marks, R. Bittman, and R. E. Pagano Inhibition of caveolar uptake, SV40 infection, and {beta}1-integrin signaling by a nonnatural glycosphingolipid stereoisomer J. Cell Biol., March 26, 2007; 176(7): 895 - 901. [Abstract] [Full Text] [PDF]