[en] Lecithin cholesterol acyltransferase (LCAT) is an interfacial enzyme active on
both high-density (HDL) and low-density lipoproteins (LDL). Threading alignments
of LCAT with lipases suggest that residues 50-74 form an interfacial recognition
site and this hypothesis was tested by site-directed mutagenesis. The
(delta56-68) deletion mutant had no activity on any substrate. Substitution of
W61 with F, Y, L or G suggested that an aromatic residue is required for full
enzymatic activity. The activity of the W61F and W61Y mutants was retained on HDL
but decreased on LDL, possibly owing to impaired accessibility to the LDL lipid
substrate. The decreased activity of the single R52A and K53A mutants on HDL and
LDL and the severer effect of the double mutation suggested that these conserved
residues contribute to the folding of the LCAT lid. The membrane-destabilizing
properties of the LCAT 56-68 helical segment were demonstrated using the
corresponding synthetic peptide. An M65N-N66M substitution decreased both the
fusogenic properties of the peptide and the activity of the mutant enzyme on all
substrates. These results suggest that the putative interfacial recognition
domain of LCAT plays an important role in regulating the interaction of the
enzyme with its organized lipoprotein substrates.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Peelman, F.
Vanloo, B.
Perez-Mendez, O.
Decout, A.
Verschelde, Jl.
Labeur, C.
Vinaimont, N.
Verhee, A.
Duverger, N.
Brasseur, Robert ; Université de Liège - ULiège > Gembloux Agro-Bio Tech
Dugi, K.A., Dichek, H.L. and Santamarina-Fojo, S. (1995) J. Biol. Chem., 270, 25396-25401.
Egloff, M-P, Marguet, F., Buono, G., Verger, R., Cambillau, C. and van Tilbeurgh, H. (1995) Biochemistry, 34, 2751-2762.
Eisenberg, D., Weiss, R.M. and Terwilliger, W.C. (1982) Nature, 299, 371-374.
Feaster, S.R., Quinn, D.M. and Barnett, B.L. (1997) Protein Sci., 6, 73-79.
Francone, O.L. and Fielding, C.J. (1991) Biochemistry, 30, 10074-10077.
Glomset, J.A. (1968) J. Lipid Res., 9, 155-167.
Grochulski, P., Li, Y., Schrag, J.D., Bouthillier, F. Smith, P., Harrison, D., Rubin, B. and Cygler, M. (1993) J. Biol. Chem., 268, 12843-12847.
Grochulski, P., Li, Y., Schrag, J.D. and Cygler, M. (1994) Protein Sci., 3, 82-91.
Hjorth, A. et al. (1993) Biochemistry, 32, 4702-4707.
Ibdah, J.A., Lund-Katz, S. and Phillips, M. (1989) Biochemistry, 28, 1126-1133.
Kuivenhoven, J.A., Pritchard, H., Frohlich, J., Assman, G. and Kastelein, J. (1997) J. Lipid Res., 38, 191-205.
Kyu Kim, K., Kyu Song, H., Hae Shin, D., Yeon Hwang, K. and Won Suh, S. (1997) Structure, 5, 173-185.
Lamhert, G. et al. (1998) Eur. J. Biochem., 253, 328-338.
Martinelle, M., Holmquist, M., Clausen, I.G., Palkar, S., Svenden, A. and Hult, K. (1996) Protein Engng, 9, 519-524.
Matz, C.E. and Jonas, A. (1982) J. Biol. Chem. 257, 4535-4540.
Ollis, D.L. et al. (1992) Protein Engng. 5, 197-211.
Peelman, F. Goethals, M., Vanloo, B., Brasseur, R., Vanderckhove, J. and Rosseneu, M. Eur. J. Biochem. (1997) 249, 708-715.
Peelman, F. et al. (1998a) Protein Sci., 7, 587-599.
Peelman, F., Verschelde, J.-L., Vanloo, B., Ampe, C., Labeur, C., Tavernier, J., Vandekerckhove, J. and Rosseneu, M (1998b). J. Lipid Res., in press.
Perez-Mendez, O., Vanloo, B., Decout, A., Goethals, M., Peelman, F., Vandekerckhove, J., Brasseur, R. and Rosseneu, M. (1998) Eur. J. Biochem., 256, 570-579.
Pillot, T., Goethals, M., Vanloo, B., Talussot, C., Brasseur, R., Vandekerckhove, J., Rosseneu, M. and Lins, L. (1996) J. Biol. Chem., 271, 28757-28765.
Rafalski, M., Ortiz, A., Rockwell, A., van Ginkel, L.C., Lear, J.D., DeGrado, W.F. and Wilschut, J. (1991) Biochemistry, 30, 10211-10220.
Schrag, J.D. and Cygler, M. (1993) J. Mol. Biol., 230, 575-591.
Soldatova, L., Kochoumian, L. and King, T.P. (1993) FEBS Lett., 320, 145-149.
Stokke, K.T. and Norum, K.R. (1971) Scand. J. Clin. Lab. Invest., 27, 21-27.
Thirstrup, K., Verger, R. and Carriere, F. (1994) Biochemistry, 33, 2748-2756.
Vanloo, B., Taveirne, J., Baert, G., Lorent, G., Vercaemst, R., Lins, L., Ruysschaert, J.M. and Rosseneu, M. (1992) Biochim. Biophys. Acta. 1128, 258-266.
Van Tilbeurgh, H, Egloff, M-P., Martinez, C., Rugani, N., Verger, R. and Cambillau, C. (1993) Nature, 362, 814-820.
Vercaemst, R., Union, A. and Rosseneu., M. (1989) J. Chromatogr., 494, 43-52.
Wang, J., De Lozier, J.A., Gebre, A.K., Dolphin, P.J. and Parks, J.S. (1998) J. Lipid Res., 39, 51-58.