Biomaterial Database

Site-directed mutagenesis of a putative heparin binding domain of avian lipoprotein lipase. 1993

D E Berryman, and A Bensadoun

Division of Nutritional Sciences, Cornell University, Ithaca, New York 14853.

Lipoprotein lipase (LPL) binds to heparin and heparan sulfate proteoglycans. We have employed site-directed mutagenesis to dissect one of the proposed heparin binding domains of avian LPL, which contains the sequence Arg-Lys-Asn-Arg (amino acids 281-284). Various single, double, and triple mutants of chicken LPL were constructed in order to alter the positive charge of this region. The mutant and wild-type cDNAs were subcloned into an expression vector, pRc/CMV, and expressed in Chinese hamster ovary cells. In general, the LPL mutants with a decrease in regional positive charge showed a decrease in affinity for heparin and heparan sulfate proteoglycans. The greatest effect was seen with the triple mutant, LPL 5G, in which all of the positively charged amino acids were altered to neutral residues. On a heparin-Sepharose column, LPL 5G eluted at 0.96 M NaCl compared with 1.35 M for wild-type LPL. This mutant also had the lowest specific activity with 1.5 mu eq fatty acid/micrograms/h for the cell-associated pool and with no detectable activity in the media. Wild-type cells, however, produced a lipase with a specific activity of 12.4 and 13.1 mu eq fatty acid/micrograms/h for cell-associated and media lipase pools, respectively. LPL 5G also showed a decrease in affinity for the heparan sulfate proteoglycans on the cell surface of Chinese hamster ovary cells. In conclusion, the region of avian LPL between Arg281 and Arg284 does appear to be involved in heparin-binding; however, additional regions must be involved since binding was not completely abolished. In addition, specific activity of the cell-associated and secreted LPL is correlated to affinity of the enzyme for heparan sulfate chains.

UI	MeSH Term	Description	Entries
D008071	Lipoprotein Lipase	An enzyme of the hydrolase class that catalyzes the reaction of triacylglycerol and water to yield diacylglycerol and a fatty acid anion. The enzyme hydrolyzes triacylglycerols in chylomicrons, very-low-density lipoproteins, low-density lipoproteins, and diacylglycerols. It occurs on capillary endothelial surfaces, especially in mammary, muscle, and adipose tissue. Genetic deficiency of the enzyme causes familial hyperlipoproteinemia Type I. (Dorland, 27th ed) EC 3.1.1.34.	Heparin-Clearing Factor,Lipemia-Clearing Factor,Diacylglycerol Lipase,Diglyceride Lipase,Post-Heparin Lipase,Postheparin Lipase,Postheparin Lipoprotein Lipase,Factor, Heparin-Clearing,Factor, Lipemia-Clearing,Heparin Clearing Factor,Lipase, Diacylglycerol,Lipase, Diglyceride,Lipase, Lipoprotein,Lipase, Post-Heparin,Lipase, Postheparin,Lipase, Postheparin Lipoprotein,Lipemia Clearing Factor,Lipoprotein Lipase, Postheparin,Post Heparin Lipase
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D009838	Oligodeoxyribonucleotides	A group of deoxyribonucleotides (up to 12) in which the phosphate residues of each deoxyribonucleotide act as bridges in forming diester linkages between the deoxyribose moieties.	Oligodeoxynucleotide,Oligodeoxyribonucleotide,Oligodeoxynucleotides
D011509	Proteoglycans	Glycoproteins which have a very high polysaccharide content.	Proteoglycan,Proteoglycan Type H
D011994	Recombinant Proteins	Proteins prepared by recombinant DNA technology.	Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D002462	Cell Membrane	The lipid- and protein-containing, selectively permeable membrane that surrounds the cytoplasm in prokaryotic and eukaryotic cells.	Plasma Membrane,Cytoplasmic Membrane,Cell Membranes,Cytoplasmic Membranes,Membrane, Cell,Membrane, Cytoplasmic,Membrane, Plasma,Membranes, Cell,Membranes, Cytoplasmic,Membranes, Plasma,Plasma Membranes
D002645	Chickens	Common name for the species Gallus gallus, the domestic fowl, in the family Phasianidae, order GALLIFORMES. It is descended from the red jungle fowl of SOUTHEAST ASIA.	Gallus gallus,Gallus domesticus,Gallus gallus domesticus,Chicken
D002846	Chromatography, Affinity	A chromatographic technique that utilizes the ability of biological molecules, often ANTIBODIES, to bind to certain ligands specifically and reversibly. It is used in protein biochemistry. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed)	Chromatography, Bioaffinity,Immunochromatography,Affinity Chromatography,Bioaffinity Chromatography
D003001	Cloning, Molecular	The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells.	Molecular Cloning
D006224	Cricetinae	A subfamily in the family MURIDAE, comprising the hamsters. Four of the more common genera are Cricetus, CRICETULUS; MESOCRICETUS; and PHODOPUS.	Cricetus,Hamsters,Hamster