HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lookene, A. Articles by Bruin, T. Search for Related Content PubMed PubMed Citation Articles by Lookene, A. Articles by Bruin, T. Social Bookmarking                      What's this?

Volume 272, Number 2, Issue of January 10, 1997 pp. 766-772
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

---

Mutation of Tryptophan Residues in Lipoprotein Lipase
EFFECTS ON STABILITY, IMMUNOREACTIVITY, AND CATALYTIC PROPERTIES

(Received for publication, August 6, 1996, and in revised form, September 22, 1996)

Aivar Lookene , Niels B. Groot ^§ , John J. P. Kastelein ^§ , Gunilla Olivecrona and Taco Bruin ^§

From the  Department of Medical Biochemistry and Biophysics, Umeå University, S-901 87 Umeå, Sweden and ^§ Center for Thrombosis, Hemostasis, Atherosclerosis and Inflammation Research, Academic Medical Centre, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands

Previous studies had pointed to an important function of a putative exposed loop in the C-terminal domain of lipoprotein lipase for activity against emulsified lipid substrates. This loop contains 3 tryptophan residues (Trp³⁹⁰, Trp³⁹³, and Trp³⁹⁴). We have expressed and characterized lipase mutants with tryptophan to alanine substitutions at positions 55, 114, 382, 390, 393, and 394 and a double mutant at residues 393 and 394. The substitutions in the N-terminal domain (W55A and W114A) led to poor expression of completely inactive lipase variants. Heparin-Sepharose chromatography showed that mutant W114A eluted at the same salt concentration as inactive wild-type monomers, indicating that this substitution prevented subunit interaction or led to an unstable dimer. In contrast, all mutants in the C-terminal domain were expressed as mixtures of monomers and dimers similarly to the wild-type. The dimers displayed at least some catalytic activity and had the same apparent heparin affinity as the active wild-type dimers. The mutants W390A, W393A, W394A, and W393A/W394A had decreased reactivity with the monoclonal antibody 5D2, indicating that the 5D2 epitope is longer than was reported earlier, or that conformational changes affecting the epitope had occurred.

The mutants W390A, W393A, W394A, and W393A/W394A had decreased catalytic activity against a synthetic lipid emulsion of long-chain triacylglycerols (Intralipid^R) and in particular against rat lymph chylomicrons. The most pronounced decrease of activity was found for the double mutant W393A/W394A which retained only 6% of the activity of the wild-type lipase, while 70% of the activity against water-soluble tributyrylglycerol was retained. In the case of chylomicrons also the affinity for the substrate particles was lowered, as indicated by severalfold higher apparent K_m values. This effect was less prominent with the synthetic lipid emulsion.

We conclude that the tryptophan cluster Trp³⁹⁰-Trp³⁹³-Trp³⁹⁴ contributes to binding of lipoprotein lipase to lipid/water interfaces. Utilizing different lipid substrates in different physical states, we have demonstrated that the tryptophan residues in the C-terminal domain may have a role also in the productive orientation of the enzyme at the lipid/water interface.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				A J Hooper, G M Crawford, J M Brisbane, K Robertson, G F Watts, F M van Bockxmeer, and J R Burnett Familial lipoprotein lipase deficiency caused by known (G188E) and novel (W394X) LPL gene mutations Ann Clin Biochem, January 1, 2008; 45(1): 102 - 105. [Abstract] [Full Text] [PDF]

				Q. Deng, J.-w. Zhai, M.-L. Michel, J. Zhang, J. Qin, Y.-y. Kong, X.-x. Zhang, A. Budkowska, P. Tiollais, Y. Wang, et al. Identification and Characterization of Peptides That Interact with Hepatitis B Virus via the Putative Receptor Binding Site J. Virol., April 15, 2007; 81(8): 4244 - 4254. [Abstract] [Full Text] [PDF]

				N. Griffon, E. C. Budreck, C. J. Long, U. C. Broedl, D. H. L. Marchadier, J. M. Glick, and D. J. Rader Substrate specificity of lipoprotein lipase and endothelial lipase: studies of lid chimeras J. Lipid Res., August 1, 2006; 47(8): 1803 - 1811. [Abstract] [Full Text] [PDF]

				A. B. Freie, F. Ferrato, F. Carriere, and M. E. Lowe Val-407 and Ile-408 in the beta5'-Loop of Pancreatic Lipase Mediate Lipase-Colipase Interactions in the Presence of Bile Salt Micelles J. Biol. Chem., March 24, 2006; 281(12): 7793 - 7800. [Abstract] [Full Text] [PDF]

				L. Zhang, G. Wu, C. G. Tate, A. Lookene, and G. Olivecrona Calreticulin Promotes Folding/Dimerization of Human Lipoprotein Lipase Expressed in Insect Cells (Sf21) J. Biol. Chem., August 1, 2003; 278(31): 29344 - 29351. [Abstract] [Full Text] [PDF]

				M. E. Lowe The triglyceride lipases of the pancreas J. Lipid Res., December 1, 2002; 43(12): 2007 - 2016. [Abstract] [Full Text] [PDF]

				H. Wong and M. C. Schotz The lipase gene family J. Lipid Res., July 1, 2002; 43(7): 993 - 999. [Abstract] [Full Text] [PDF]

				J. Feng, H. Wehbi, and M. F. Roberts Role of Tryptophan Residues in Interfacial Binding of Phosphatidylinositol-specific Phospholipase C J. Biol. Chem., May 24, 2002; 277(22): 19867 - 19875. [Abstract] [Full Text] [PDF]

				O. Ben-Zeev, H. Z. Mao, and M. H. Doolittle Maturation of Lipoprotein Lipase in the Endoplasmic Reticulum. CONCURRENT FORMATION OF FUNCTIONAL DIMERS AND INACTIVE AGGREGATES J. Biol. Chem., March 15, 2002; 277(12): 10727 - 10738. [Abstract] [Full Text] [PDF]

				M. O. Pentikainen, R. Oksjoki, K. Oorni, and P. T. Kovanen Lipoprotein Lipase in the Arterial Wall: Linking LDL to the Arterial Extracellular Matrix and Much More Arterioscler. Thromb. Vasc. Biol., February 1, 2002; 22(2): 211 - 217. [Abstract] [Full Text] [PDF]

				H. Laurell, J. A. Contreras, I. Castan, D. Langin, and C. Holm Analysis of the psychrotolerant property of hormone-sensitive lipase through site-directed mutagenesis Protein Eng. Des. Sel., October 1, 2000; 13(10): 711 - 717. [Abstract] [Full Text] [PDF]

				V. Briquet-Laugier, O. Ben-Zeev, A. White, and M. H. Doolittle cld and lec23 are disparate mutations that affect maturation of lipoprotein lipase in the endoplasmic reticulum J. Lipid Res., November 1, 1999; 40(11): 2044 - 2058. [Abstract] [Full Text]

				S.-F. Chang, B. Reich, J. D. Brunzell, and H. Will Detailed characterization of the binding site of the lipoprotein lipase-specific monoclonal antibody 5D2 J. Lipid Res., December 1, 1998; 39(12): 2350 - 2359. [Abstract] [Full Text]

				J. S. Hill, D. Yang, J. Nikazy, L. K. Curtiss, J. T. Sparrow, and H. Wong Subdomain Chimeras of Hepatic Lipase and Lipoprotein Lipase. LOCALIZATION OF HEPARIN AND COFACTOR BINDING J. Biol. Chem., November 20, 1998; 273(47): 30979 - 30984. [Abstract] [Full Text] [PDF]

				R. Buscà, M. Martínez, E. Vilella, J. Peinado, J. L. Gelpi, S. Deeb, J. Auwerx, M. Reina, and S. Vilaró The carboxy-terminal region of human lipoprotein lipase is necessary for its exit from the endoplasmic reticulum J. Lipid Res., April 1, 1998; 39(4): 821 - 833. [Abstract] [Full Text]

				C. Desrumaux, C. Labeur, A. Verhee, J. Tavernier, J. Vandekerckhove, M. Rosseneu, and F. Peelman A Hydrophobic Cluster at the Surface of the Human Plasma Phospholipid Transfer Protein Is Critical for Activity on High Density Lipoproteins J. Biol. Chem., February 16, 2001; 276(8): 5908 - 5915. [Abstract] [Full Text] [PDF]