Journal of Lipid Research, Vol 34, 1393-1340, Copyright © 1993 by Lipid Research, Inc.

ARTICLES

Functional characterization of a chimeric lipase genetically engineered from human lipoprotein lipase and human hepatic lipase

HL Dichek, C Parrott, R Ronan, JD Brunzell, HB Brewer Jr and S Santamarina-Fojo
Molecular Disease Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892.

Lipoprotein lipase (LPL) and hepatic lipase (HL) mediate the hydrolysis of triglycerides and phospholipids present in circulating lipoprotein particles and are essential for normal lipid metabolism. Both enzymes have a similar primary amino acid structure and share requirements for intact catalytic, lipid binding, and heparin binding domains. However, LPL and HL exhibit different substrate specificities and cofactor requirements. In order to characterize the functional domains necessary for LPL activity, a chimeric lipase consisting of the amino-terminal 314 amino acids of human LPL and the carboxyl-terminal 146 amino acids of human HL was synthesized by joining the cDNA of both lipases at the 5'-end of exon 7. Northern blot hybridization and Western blot analyses revealed the size of the chimera mRNA and protein to be approximately 1.5 kb and 55 kDa, respectively. The chimeric enzyme hydrolyzed both long chain and short chain fatty acid triacylglycerols and had catalytic properties that were similar to lipoprotein lipase. Thus, apolipoprotein (apo)C-II was required for maximal lipase activity, and high salt concentration abolished the ability of the chimera to hydrolyze triolein even in the presence of apoC-II. A monospecific anti- HL polyclonal antibody interacting with the C-terminal HL-derived domain of the chimeric enzyme abolished the enzyme's ability to hydrolyze triglyceride emulsion but not tributyrin substrates. Analysis of the heparin binding properties of the chimeric enzyme using heparin- Sepharose affinity chromatography revealed an elution pattern which was intermediate between that of lipoprotein and hepatic lipase. In summary, we have characterized the functional properties of an LPL-HL chimeric enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				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]

				Y. Kojma, K.-i. Hirata, T. Ishida, Y. Shimokawa, N. Inoue, S. Kawashima, T. Quertermous, and M. Yokoyama Endothelial Lipase Modulates Monocyte Adhesion to the Vessel Wall: A POTENTIAL ROLE IN INFLAMMATION J. Biol. Chem., December 24, 2004; 279(52): 54032 - 54038. [Abstract] [Full Text] [PDF]

				R. J. Brown, A. Gauthier, R. J. Parks, R. McPherson, D. L. Sparks, J. R. Schultz, and Z. Yao Severe Hypoalphalipoproteinemia in Mice Expressing Human Hepatic Lipase Deficient in Binding to Heparan Sulfate Proteoglycan J. Biol. Chem., October 8, 2004; 279(41): 42403 - 42409. [Abstract] [Full Text] [PDF]

				J. Lopez-Miranda, G. Cruz, P. Gomez, C. MarIn, E. Paz, P. Perez-MartInez, F. J. Fuentes, J. M. Ordovas, and F. Perez-Jimenez The Influence of Lipoprotein Lipase Gene Variation on Postprandial Lipoprotein Metabolism J. Clin. Endocrinol. Metab., September 1, 2004; 89(9): 4721 - 4728. [Abstract] [Full Text] [PDF]

				R. J. Brown, J. R. Schultz, K. W. S. Ko, J. S. Hill, T. A. Ramsamy, A. L. White, D. L. Sparks, and Z. Yao The amino acid sequences of the carboxyl termini of human and mouse hepatic lipase influence cell surface association J. Lipid Res., July 1, 2003; 44(7): 1306 - 1314. [Abstract] [Full Text] [PDF]

				T. L. McIlhargey, Y. Yang, H. Wong, and J. S. Hill Identification of a Lipoprotein Lipase Cofactor-binding Site by Chemical Cross-linking and Transfer of Apolipoprotein C-II-responsive Lipolysis from Lipoprotein Lipase to Hepatic Lipase J. Biol. Chem., June 13, 2003; 278(25): 23027 - 23035. [Abstract] [Full Text] [PDF]

				J. Peterson, A. F. Ayyobi, Y. Ma, H. Henderson, M. Reina, S. S. Deeb, S. Santamarina-Fojo, M. R. Hayden, and J. D. Brunzell Structural and functional consequences of missense mutations in exon 5 of the lipoprotein lipase gene J. Lipid Res., March 1, 2002; 43(3): 398 - 406. [Abstract] [Full Text] [PDF]

				Y. Shen, A. Lookene, S. Nilsson, and G. Olivecrona Functional Analyses of Human Apolipoprotein CII by Site-directed Mutagenesis. IDENTIFICATION OF RESIDUES IMPORTANT FOR ACTIVATION OF LIPOPROTEIN LIPASE J. Biol. Chem., February 1, 2002; 277(6): 4334 - 4342. [Abstract] [Full Text] [PDF]

				J. C. Boedeker, M. H. Doolittle, and A. L. White Differential effect of combined lipase deficiency (cld/cld) on human hepatic lipase and lipoprotein lipase secretion J. Lipid Res., November 1, 2001; 42(11): 1858 - 1864. [Abstract] [Full Text] [PDF]

				R. A. Sendak, D. E. Berryman, G. Gellman, K. Melford, and A. Bensadoun Binding of hepatic lipase to heparin: identification of specific heparin-binding residues in two distinct positive charge clusters J. Lipid Res., February 1, 2000; 41(2): 260 - 268. [Abstract] [Full Text]

				J. C. Boedeker, M. Doolittle, S. Santamarina-Fojo, and A. L. White Role of N-linked carbohydrate processing and calnexin in human hepatic lipase secretion J. Lipid Res., September 1, 1999; 40(9): 1627 - 1635. [Abstract] [Full Text]

				G. Lambert, M. B. Chase, K. Dugi, A. Bensadoun, H. B. Brewer , Jr., and S. Santamarina-Fojo Hepatic lipase promotes the selective uptake of high density lipoprotein-cholesteryl esters via the scavenger receptor B1 J. Lipid Res., July 1, 1999; 40(7): 1294 - 1303. [Abstract] [Full Text]

				K.-i. Hirata, H. L. Dichek, J. A. Cioffi, S. Y. Choi, N. J. Leeper, L. Quintana, G. S. Kronmal, A. D. Cooper, and T. Quertermous Cloning of a Unique Lipase from Endothelial Cells Extends the Lipase Gene Family J. Biol. Chem., May 14, 1999; 274(20): 14170 - 14175. [Abstract] [Full Text] [PDF]

				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. A. Sendak and A. Bensadoun Identification of a heparin-binding domain in the distal carboxyl-terminal region of lipoprotein lipase by site-directed mutagenesis J. Lipid Res., June 1, 1998; 39(6): 1310 - 1315. [Abstract] [Full Text]

				Z.-S. Ji, H. L. Dichek, R. D. Miranda, and R. W. Mahley Heparan Sulfate Proteoglycans Participate in Hepatic Lipaseand Apolipoprotein E-mediated Binding and Uptake of Plasma Lipoproteins, Including High Density Lipoproteins J. Biol. Chem., December 12, 1997; 272(50): 31285 - 31292. [Abstract] [Full Text] [PDF]

				M. S. Nielsen, J. Brejning, R. Garcia, H. Zhang, M. R. Hayden, S. Vilaro, and J. Gliemann Segments in the C-terminal Folding Domain of Lipoprotein Lipase Important for Binding to the Low Density Lipoprotein Receptor-related Protein and to Heparan Sulfate Proteoglycans J. Biol. Chem., February 28, 1997; 272(9): 5821 - 5827. [Abstract] [Full Text] [PDF]

				A. Lookene, N. B. Groot, J. J.P. Kastelein, G. Olivecrona, and T. Bruin Mutation of Tryptophan Residues in Lipoprotein Lipase. EFFECTS ON STABILITY, IMMUNOREACTIVITY, AND CATALYTIC PROPERTIES J. Biol. Chem., January 10, 1997; 272(2): 766 - 772. [Abstract] [Full Text] [PDF]

				J. Kobayashi, D. Applebaum-Bowden, K. A. Dugi, D. R. Brown, V. S. Kashyap, C. Parrott, C. Duarte, N. Maeda, and S. Santamarina-Fojo Analysis of Protein Structure-Function in Vivo. ADENOVIRUS-MEDIATED TRANSFER OF LIPASE LID MUTANTS IN HEPATIC LIPASE-DEFICIENT MICE J. Biol. Chem., October 18, 1996; 271(42): 26296 - 26301. [Abstract] [Full Text] [PDF]

				J. S. Hill, R. C. Davis, D. Yang, J. Wen, J. S. Philo, P. H. Poon, M. L. Phillips, E. S. Kempner, and H. Wong Human Hepatic Lipase Subunit Structure Determination J. Biol. Chem., September 13, 1996; 271(37): 22931 - 22936. [Abstract] [Full Text] [PDF]

				K. A. Dugi, H.én L. Dichek, and S. Santamarina-Fojo Human Hepatic and Lipoprotein Lipase: The Loop Covering the Catalytic Site Mediates Lipase Substrate Specificity J. Biol. Chem., October 27, 1995; 270(43): 25396 - 25401. [Abstract] [Full Text] [PDF]